New drug discovery of cardiac anti-arrhythmic drugs: insights in animal models

Cardiac rhythm regulated by micro-macroscopic structures of heart. Pacemaker abnormalities or disruptions in electrical conduction, lead to arrhythmic disorders may be benign, typical, threatening, ultimately fatal, occurs in clinical practice, patients on digitalis, anaesthesia or acute myocardial infarction. Both traditional and genetic animal models are: In-vitro: Isolated ventricular Myocytes, Guinea pig papillary muscles, Patch-Clamp Experiments, Porcine Atrial Myocytes, Guinea pig ventricular myocytes, Guinea pig papillary muscle: action potential and refractory period, Langendorff technique, Arrhythmia by acetylcholine or potassium. Acquired arrhythmia disorders: Transverse Aortic Constriction, Myocardial Ischemia, Complete Heart Block and AV Node Ablation, Chronic Tachypacing, Inflammation, Metabolic and Drug-Induced Arrhythmia. In-Vivo: Chemically induced arrhythmia: Aconitine antagonism, Digoxin-induced arrhythmia, Strophanthin/ouabain-induced arrhythmia, Adrenaline-induced arrhythmia, and Calcium-induced arrhythmia. Electrically induced arrhythmia: Ventricular fibrillation electrical threshold, Arrhythmia through programmed electrical stimulation, sudden coronary death in dogs, Exercise ventricular fibrillation. Genetic Arrhythmia: Channelopathies, Calcium Release Deficiency Syndrome, Long QT Syndrome, Short QT Syndrome, Brugada Syndrome. Genetic with Structural Heart Disease: Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia, Dilated Cardiomyopathy, Hypertrophic Cardiomyopathy, Atrial Fibrillation, Sick Sinus Syndrome, Atrioventricular Block, Preexcitation Syndrome. Arrhythmia in Pluripotent Stem Cell Cardiomyocytes. Conclusion: Both traditional and genetic, experimental models of cardiac arrhythmias’ characteristics and significance help in development of new antiarrhythmic drugs.

The specialized cells within the sinoatrial node, AV node, and His-Purkinje system are capable of spontaneous depolarization that is regulated by both the sympathetic and parasympathetic nervous system. Conduction through the heart depends on electrical coupling between cells, which is mediated by gap junctions. Species Differences in the Cardiac Action Potential and Cardiac Ca2 + Handling The cardiac action potential (AP) results from the opening and closing of ion channels and electrogenic transporters in the plasma membrane of individual cardiomyocytes (see study by Varró et al. for details) 15 . Figure 2 illustrates AP wave forms and underlying  The ventricular action potential and ionic currents in humans and mice. Note the differences in action potential shape, which is caused primarily by differences in ionic currents circled in red. I Ca(L) indicates L-type Ca current; I Ca(T) , T-type Ca current; I Na , Na current; I NaCa , Na-Ca-Exchange current; I NaK , NaK-ATPase pump current; I K1 , Inward rectifier K-current; I Kr , rapidly activating delayed rectifier K-current; I Ks , slowly activating delayed rectifier K-current; I ss , rapidly activating steady-state K-currents; and I to , transient outward K-current. Please note that current densities (pA/pF) measured in single cells vary drastically with experimental conditions and voltage clamp protocols. Current densities were chosen to reflect relative contributions to the AP.

In-vitro models
Isolated guinea pig papillary muscles A fast and accurate method without microelectrodes is available for identifying and classifying potential antiarrhythmic drugs into Classes I, II, III and IV. In the guinea pig right ventricle, papillary muscle excitability, developed tone (DT) and effective refractory period (ERP) are evaluated. Inhibition of the Na + channel decreases excitability, inhibition of the K + channel increases refractory period, and blockade of the Ca2 + channel decreases myocardial tone.
Class I antiarrhythmic agents are heterogeneous with respect to their cardiac electrophysiological effects and have been subdivided into three categories: la, lb and lc. The purpose of the present study was to determine the classification and investigate the mechanism of action of ACC-9358 [4-hydroxy-N-phenyl-3,5-bis (1-pyrrolidinyl-methyl)benzamide], a novel class I antiarrhythmic agent currently under clinical investigation. The effects of ACC-9358 on action potentials from isolated canine Purkinje fibers and ventricular muscle were examined using standard microelectrode techniques. In Purkinje fibers, ACC-9358 (1-50 microM) exerted a dose-dependent reduction in maximum upstroke velocity (Vmax) and action potential duration at 50 and 90% repolarization (APD50 and APD90). The reduction of Vmax was voltage-dependent (greater at an extracellular potassium concentration of 6 mM than at 2.7 mM), frequency-dependent (greater at a basic cycle length of 500 than at 2000 ms) and very slow in onset (rate constant of 0.017 action potentials-1) and offset (recovery half-time of 66.9 s). In Purkinje fibers, ACC-9358 attenuated the action potential shortening effects of lidocaine but not that of nicardipine or nicorandil and shortened APD50 to a greater extent at a basic cycle length of 2000 than at 500 ms. In ventricular muscle, ACC-9358 (1-50 microM) exerted a dose-dependent reduction in Vmax and prolongation of APD50 and APD90 31 . Figure 4 is a diagrammatic illustration of the preparation. The auricles are dissected from the heart of a rabbit and suspended in a bath of oxygenated Ringer-Locke at 29 °C.; at their upper end they are fixed in a pair of platinum electrodes just above the surface of the bath. The sharpened tips of the electrodes project into a tiny chamber at the bottom of a perspex rod. This chamber tapers at its lower end to form an oval opening; a silk thread is tied through the tip of one auricle, which is then drawn into the chamber so that the electrodes penetrate its substance, and so that the auricle itself completely seals the oval opening. At its upper end the chamber is continuous with a circular channel which runs through the perspex rod; a gentle current of warm air is blown down this channel at constant pressure, and serves the double purpose of oxygenating the tissue in the chamber and of preventing Ringer-Locke entering the chamber by capillary attraction, and so shortcircuiting the electrodes. The object of this device is to ensure that, while the main part of the muscle is immersed in the bath, the electrodes are outside it; when methyl violet was added to the bath, the tip of the auricle drawn up into the electrode chamber was scarcely stained at all. The contraction of the muscle is recorded by a lever writing on a smoked drum, and attached to the lower end of the auricles by a silk thread running round a pulley immersed in the bath. The auricles contract spontaneously (at a rate of 80-120 per minute), and they can also be stimulated by break-shocks from an induction coil at any desired speed, using a Lewis rotary contact-breaker. The coil separation is adjusted so that the peak voltage in the secondary is about ten times that necessary to cause extrasystoles at the beginning of the experiment; this ensures that stimuli are so far above threshold that the notorious irregularity of induction shocks will not be of practical significance. As the rate of electrical stimulation is increased the auricle follows each stimulus up to a certain point (between 250 and 350 per minute) at which it begins to drop beats (because the interval between shocks is less than the absolute refractory period; cf. Mines, 1913). It is easy to distinguish these dropped beats since the next auricular contraction is more powerful. Thus in t experiment shown in Fig. 2 the auricle followed each stimulus at 200 and 260 per minute to 266 per minute it dropped a single beat, and at 274 per minute the response soon became ery irregular; at 338 per minute it had adopted a 2: 1 rhythm. In this instance the maximal rate at which the auricle could respond would be recorded as 260 per minute. The method is based upon the observation that quinidine reduces this maximal rate 32 .
The maximal rate at which the auricle responds to electrical stimuli is 218/min.; after quinidine 1:100,000 for 10 min this is reduced to 184/min. i.e., the maximal rate was reduced by 34 per minute or 15.6 per cent 32 .
Aftercontractions, delayed afterdepolarizations, and automaticity occurred in guinea pig papillary muscles that were reoxygenated after hypoxic conditioning. The emergence of dysfunction was dependent on the severity of hypoxic conditioning and on stimulation during reoxygenation. After 60 min of substrate-free hypoxia, reoxygenation induced automaticity in a high proportion of stimu-lated muscles; the automaticity appeared within 1 min and lasted for 10-20 min. After similar conditioning, muscles reoxygenated for 7-15 min were stimulated at various cycle lengths. The incidence of automaticity and the amplitudes of delayed events had www.nature.com/scientificreports/ The novel compound AVE1231 was investigated in order to elucidate its potential against atrial fibrillation. In CHO cells, the current generated by hKv1.5 or hKv4.3 + KChIP2.2b channels was blocked with IC50 values of 3.6 microM and 5.9 microM, respectively. In pig left atrial myocytes, a voltage-dependent outward current was blocked with an IC50 of 1.1 microM, mainly by accelerating the time constant of decay. Carbachol-activated IKACh was blocked by AVE1231 with an IC50 of 8.4 microM. Other ionic currents, like the IKr, IKs, IKATP, ICa, and INa were only mildly affected by 10 microM AVE1231. In guinea pig papillary muscle the APD90 and the upstroke velocity were not significantly altered by 30 microM AVE1231. In anesthetized pigs, oral doses of 0.3, 1, and 3 mg/kg AVE1231 caused a dose-dependent increase in left atrial refractoriness (LAERP), associated by inhibition of left atrial vulnerability to arrhythmia. There were no effects on the ECG intervals, ventricular monophasic action potentials, or ventricular refractory periods at 3 mg/kg AVE1231 applied intravenously. In conscious goats, both AVE1231 (3 mg/kg/h iv) and dofetilide (10 microg/kg/h iv) significantly prolonged LAERP. After 72 h of tachypacing, when LAERP was shortened significantly (electrical remodelling), the prolongation of LAERP induced by AVE1231 was even more pronounced than in sinus rhythm. In contrast, the effect of dofetilide was strongly decreased. The present data demonstrate that AVE1231 blocks early atrial K channels and prolongs atrial refractoriness with no effects on ECG intervals and ventricular repolarisation, suggesting that it is suited for the prevention of atrial fibrillation in patients 34 .
Limitations of Patch-Clamp Experiments in CHO Cells used to assess ventricular arrhythmias but not Atrial Fibrillation-Flutter & Torsades de Pointes Arrhythmias.

Isolation of porcine atrial myocytes
All investigations with animals conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-13, revised 1996) and were performed following approval by the Ethical Review Board of the State of Hessen and in accordance with the German animal protection law.
Male pigs (German Landrace) weighing 15 to 30 kg were anesthetized with pentobarbital as described previously. After a left thoracotomy, the lung was retracted, the pericardium incised, and the heart quickly removed and placed in oxygenated, nominally Ca 2+ -free Tyrode solution containing (in mM): NaCl 143, KCl 5.4, MgCl 2 0.5, NaH 2 PO 4 0.25, HEPES 5, and glucose 10, pH adjusted to 7.2 with NaOH. The hearts were then mounted on a Langendorff apparatus and perfused via the left circumflex coronary artery with Tyrode solution at 37 °C with constant pressure (80 cm H 2 O). All coronary vessels descending to the ventricular walls were ligated, ensuring sufficient perfusion of the left atrium. When the atrium was clear of blood and contraction had ceased (∼5 min), perfusion was continued with the same Tyrode solution, which now contained 0.015 mM CaCl 2 and 0.03% collagenase (type CLS II, Biochrom KG, Berlin, Germany), until atrial tissue softened (∼20 min). Thereafter, left atrial tissue was cut into small pieces and mechanically dissociated by trituration. Cells were then washed with storage solution containing (in mM): L-glutamic acid 50, KCl 40, taurine 20, KH 2 PO 4 20, MgCl 2 1, glucose 10, HEPES 10, EGTA 2 (pH 7.2 with KOH), and then filtered through a nylon mesh. The isolated cells were kept at room temperature in the storage solution 35 .
Limitations of Isolation of Porcine Atrial Myocytes used to assess Atrial Fibrillation-Flutter arrhythmias but not ventricular Arrhythmias.
Atrial fibrillation (AF) is the most common sustained arrhythmia encountered in humans and is a significant source of morbidity and mortality. Despite its prevalence, our mechanistic understanding is incomplete, the therapeutic options have limited efficacy, and are often fraught with risks. A better biological understanding of AF is needed to spearhead novel therapeutic avenues. Although "natural" AF is nearly nonexistent in most species, animal models have contributed significantly to our understanding of AF and some therapeutic options. However, the impediments of animal models are also apparent and stem largely from the differences in basic physiology as well as the complexities underlying human AF; these preclude the creation of a "perfect" animal model and have obviated the translation of animal findings. Herein, we review the vast array of AF models available, spanning the mouse heart (weighing 1/1000th of a human heart) to the horse heart (10 × heavier than the human heart). We attempt to highlight the features of each model that bring value to our understanding of AF but also the shortcomings and pitfalls. Finally, we borrowed the concept of a SWOT analysis from the business community (which stands for strengths, weaknesses, opportunities, and threats) and applied this introspective type of analysis to animal models for AF. We identify unmet needs and stress that is in the context of rapidly advancing technologies, these present opportunities for the future use of animal models 36 .
Isolation of guinea pig ventricular myocytes Ventricular myocytes were isolated by enzymatic digestion according to the same procedure as above. We used 400 g Dunkin Hardley Pirbright White guinea pigs to kill them quickly by concussion and cervical dislocation. The chest was opened; the hearts removed and immediately placed in ice-cold isotonic saline. Retrograde cardiac perfusion through the aorta at 37 °C was performed with the same solutions used for the isolation of porcine atrial myocytes 37 .
Limitations of Isolation of Guinea Pig Ventricular Myocytes used to assess ventricular arrhythmias but not Atrial Fibrillation-Flutter arrhythmias.
Isolated guinea pig papillary muscle: action potential and refractory period After electrical stimulation, the papillary muscle of the left ventricle of a guinea pig registers an intracellular action potential. The stimulation frequency is changed to determine the refractory period. The pro-or antiarrhythmic effect of a chemical can be identified by the length of the effective refractory period.The inotropic effect of the test substance is also determined.
The electromechanical effects of UK-68,798 (UK), a novel class III antiarrhythmic drug, were studied in guinea pig and rat papillary muscles (PMs) and atria in vitro using conventional microelectrode technique. UK (10(-8)-10(-6) M) prolonged the action potential duration (APD) by 21-58% and effective refractory period in parallel, without affecting the resting potential or maximum rate of depolarization in guinea pig PM stimulated at 1 Hz. UK increased the contractile force without prolonging the time to peak force or relaxation. In comparison, 5 × 10(-5) M d-sotalol was needed to induce the same electrophysiological effects as 10(-8) M UK. UK prolonged the APD significantly less at 2 Hz than at 1 and 0.5 Hz. Early afterdepolarizations (EADs) developed in 2 of 11 preparations after 10(-6) M at 0.5 Hz. No reversal of drug effect was seen after up to 2 h washout. UK (10(-9)-10(-5) M) reduced the spontaneous heart rate and prolonged the sinus node recovery time of guinea pig right atria. No effects on rat PM or atria, even after 10(-5) M, indicate a selective action of UK on the delayed rectifying outward potassium current, Ik. These results indicate a potent and selective, rate-dependent class III antiarrhythmic action of UK-68,798 linked with positive inotropy. Increased APD, bradycardia, and induction of EADs, however, represent a potential arrhythmogenic combination 38 .
Limitations of Action potential and refractory period in isolated guinea pig papillary muscle used to assess ventricular arrhythmias but not Atrial Fibrillation-Flutter & Torsades de Pointes Arrhythmias.

Langendorff technique
This technique's fundamental idea is that the heart is supplied with oxygenated saline solutions at constant pressure or flow in a retrograde route from the aorta. Similar to the in-situ heart during diastole, retrograde perfusion shuts the aortic valves. Through the coronary arteries branching off the coronary sinus and the expanded right atrium, the perfusate is displaced. By stunning, guinea pigs of either sex that weigh between 300 and 500 g are sacrificed. At 37, the heart was rapidly removed and placed in a dish with Ringer's solution. Lung and pericardial tissues that are related are taken out. Below the point of division, the aorta is located and severed. The aorta is punctured, the cannula is tied, and oxygenated Ringer's solution is then injected into the heart. The heart is moved to a double-walled perfusion device made of plexiglass that is kept at a temperature of 37 °C. At a constant pressure and temperature of 40 mm Hg and 37 °C, oxygenated Ringer's solution is perfused from a reservoir. The LAD coronary artery is bound with a ligament, and an occlusion is maintained for 10 min before reperfusion. Before or after occlusion, test chemical is given via perfusion medium. Because pulsatile stimulation and arrhythmia induction involve epicardial ECG electrodes (rectangular pulses of 0.75 ms duration, usually of 10 V; frequency 400-1800 shocks per min). At the top of the heart is a little steel hook with a rope. A force transducer measures contractile force isometrically, and the results are recorded on a polygraph. A chronometer connected to the polygraph measures heart rate. The perfusion medium is infused with drugs. Both the control group and the test group's ventricular fibrillation or ventricular tachycardia frequency and duration are noted 39 .
Increased atrial pressure in the isolated rabbit heart resulted in a significant increase in vulnerability to Atrial fibrillation (AF) that was closely correlated to shortening of the Right and left atrial effective refractory periods (AERPs). These changes were completely reversible within 3 min after release of the atrial stretch, resulting in prompt termination of AF (Figs. 5 and 6) 39 . There are no limitations Langendorff technique have vulnerability to atrial and ventricular Fibrillation-Flutter.
Acetylcholine or potassium-induced arrhythmia New Zealand white rabbits weighing between 0.5 and 3 kg were used for the experiment. The hearts of the slaughtered animals are immediately removed. In Ringer's solution, more tissue is removed from the atria. There is an electrode at the bottom of the tank to which the vestibules are connected and used. The atria are fibrillated with acetylcholine (3 × 10 g/mL) or potassium chloride (0.10 g). (Frequency: 400-1800 impacts per minute). Machines are used to create kymographic records. It takes up to 10 min to treat arrhythmias to prevent them from developing and continuing. After a 30 min inactivity period, the test chemical is added to the bath and fibrillation continues. After administration of the test material, the preparation is washed off and if the atria do not stop fibrillating within 8-10 min, normal contraction resumes.The effectiveness of the test chemical is determined by whether the fibrillation disappears immediately or within five minutes of placing the test drug in the organ bath 40,41 .
There are no limitations, Acetylcholine or potassium-induced arrhythmia, vulnerability to atrial and ventricular Fibrillation.

Animal models of acquired arrhythmia disorders
Acquired heart disease is the most common cause of increased arrhythmia risk in humans. Acquired heart disease develops over the course of a person's life becasue of structural remodeling associated with hypertension 42 , coronary artery disease 43 , nonischemic cardiomyopathy 44 , primary and secondary valvular disease 45 , autoimmune rheumatic diseases 44 , and myocarditis 46 . Acute cardiac stress or injury leads to activation of specific cell signaling pathways 47,48 , mitochondrial dysfunction 49,50 , altered Ca2 + handling [51][52][53] , and a switch in metabolism 54 from fatty acid oxidation to glycolysis 55 . This leads to chronic changes in gene expression of trophic and mitotic factors, inflammation, and ultimately to myocyte hypertrophy and fibrosis. Pathological fibrosis is characterized by excessive proliferation of cardiac fibroblasts and ECM (extracellular matrix) protein deposition. Several key profibrotic factors have been identified, including TGF (transforming growth factor)-β, angiotensin II and aldosterone, which contribute to the development of cardiac fibrosis rssegardless of the underlying pathology 56 . However, the relative contribution of a distinct molecular pathway depends on the type and the degree of the initial cardiac injury. Various animal models have been developed to study aspects of these acute and chronic changes leading to arrhythmia risk and are discussed below (Table 1) www.nature.com/scientificreports/

Transverse aortic constriction
The transverse aortic constriction (TAC) model mimics chronic hypertension or aortic stenosis by causing a stricture in the thoracic aorta 57 Left ventricular and atrial pressure overload increases the wall tension leading to hypertrophy, chamber dilation, and fibrosis 58,59 . This can be done in various methods, including sutures 57,58 , inflatable cuffs 60 or intravascular stents 59,61,62 . Within hours of injury, myocyte hypertrophy is induced by activation of p38 MAP Kinase 63 , ERK1/2 64 , and PI3K/AKT signaling 65 . Increased TGF-β production due to cytokine 66 and adrenergic receptor activation 67,68 leads to fibroblast proliferation and collagen deposition. The reninangiotensinaldosterone system plays an important role in developing hypertrophy and arrhythmia, as treatment with ACE inhibitors 69 and spironolactone 70 reduces fibrosis and improving conduction velocity. This initially leads to ventricular hypertrophy, later followed by chamber dilation 66 . While recruitment of Ly6ClowCXCR1 + macrophages has been found in the LV early after TAC 71 , there is histologically less inflammation in this model than in other cardiac injury models 64 . Notable in this model is the upregulation of NCX 72 and downregulation of SERCA2a in myocytes over time, which is also seen in explant human hearts with reduced ejection fraction. While restoring SERCA2 has shown promise in improving systolic dysfunction 73,74 and suppression of ventricular arrhythmias 75,76 in animal models, the CUPID2 trial in patients with human hearts with reduced ejection fraction showed neutral results 77 . In mice with TAC, multiple investigators have shown an increase conduction time, AP duration, and AV nodal refractory period, QT prolongation with inducible atrial (50%-60%) and ventricular arrhythmias (40%-50%), but spontaneous arrhythmias in vivo are rare [78][79][80][81] . Redistribution of connexin-43 (Cx43) laterally away from intercalating disk has been purposed to explain, in part, the changes in conduction in this model 80 .
Arrhythmia induction can be challenging in this model, which is likely due to variability in the extent of constriction postprocedure 82 , the length of time of injury before analysis, sex, and strain [83][84][85][86][87] . Care must be taken when comparing results from different injury protocols and strains in this model. Similarly, rabbits, rats, and guinea swine develop ventricular hypertrophy and dilation after TAC, albeit over a more extended period (8 versus 4 weeks in mice) 88 and with a higher incidence of sudden cardiac death. Unlike the mouse model, rabbits [89][90][91] (aortic insufficiency with abdominal aortic constriction), rats 75,79 , and guinea pigs [92][93][94] , develop spontaneous arrhythmias in response to catecholamine challenge. Similarly, these animals develop ventricular hypertrophy,  www.nature.com/scientificreports/ chamber dilation and fibrosis after TAC, albeit over a more extended period (8 versus 4 weeks in mice) 88 , and with a higher incidence of sudden cardiac death. AP prolongation is a hallmark in these models and is linked to increased INaL and INCX with reduced ICaL responsiveness to β-adrenergic stimulus and increased CaMKII activity, which is seen in human heart failure myocytes 95 . Overall, these animals seem to represent a better model for arrhythmias than mice, albeit most studies use explanted heart in the Langendorff system. Unlike small animals, no studies report an increase in arrhythmia in large animals with TAC. Ascending aortic constriction in pig and sheep has been described with polyester band 96 , or an implanted inflatable cuff 60,97 . The swine model of TAC differs from other species as they tend to develop heart failure with preserved ejection fraction characterized by LV hypertrophy with diastolic dysfunction [98][99][100][101] . In contrast to pig, sheep develop cardiac dysfunction at 6 to 18 weeks post-TAC with elevated markers of ECM remodeling, chemokine production, and apoptosis 102 , but no study reported arrhythmia generation outside procedural effects. Limitations: Severity of injury dependent on strain (BALB/c > C57BL/6 > 129S1/SvImJ) and sub-strain (C57 BL/6Tac > C57BL/6NCrl > C57BL/6 J). Model requires PES to induce in vivo arrhythmias.

Myocardial ischemia
Acute and chronic myocardial ischemia is a major cause of ventricular arrhythmias in humans. During acute ischemic, myocytes are exposed to hypoxia, acidosis, increased extracellular K + , and intracellular Ca2 + 103,104 . Under ischemic conditions, cardiomyocyte mitochondria switch to glycolysis from fatty acid oxidation to maximize ATP production with limited oxygen supply 105 . In addition, hypoxia leads to reactive oxygen species production that further damages intracellular proteins and organelles 106 . Myocardial infarction occurs with myocyte apoptosis and replacement fibrosis if normal blood flow is not restored. This is a highly inflammatory model, with significant infiltration of CD11b + macrophages and upregulation of inflammatory cytokines (CCL2, TNF-α, and IL-10) after injury, leading to proinflammatory Ly6chighCCR2 + macrophages early, then pro-wound healing Ly6clowCXCR1 + chronically, which contribute to interstitial fibrosis in the border zone 107 . Chronically, myocardial fibrosis leads to conduction heterogeneity, a substrate for reentry arrhythmias. In mice and rats, myocardial ischemia is induced surgically, either transiently by ischemia/reperfusion (I/R) injury 108,109 or complete occlusion by coronary artery ligation 108,110 or cryoinjury 109,111,112 . Unlike complete occlusion, I/R injury produces reversible ischemia that leads to significant myocardial dysfunction without widespread necrosis in the area at risk. Spontaneous ventricular arrhythmias are observed during the reperfusion phase 108 . However, spontaneous ventricular arrhythmias are rare after complete occlusion aside from isolated preventricular contractions (internal data). In Figure 6. Isolated Langendorff heart perfusion model, (  www.nature.com/scientificreports/ addition, LV dysfunction leads to volume overload in the left atrium, causing fibrosis and susceptibility to AF 113 . Atrial and ventricular arrhythmias can be induced universally in isolated explanted post-MI hearts, with the occurrence of VT (inducible in up to 90%-100% in mice), VF (inducible in up to 89% of rats), and AF (inducible in 73% of rats and 33% of mice) [114][115][116][117][118][119][120] . In vivo ventricular arrhythmia induction is more difficult, requiring rapid pacing protocols [121][122][123] and a catecholamine challenge. Transvenous, pericardial, and transesophageal protocols have been described for AF and ventricular arrhythmia induction, with the occurrence of VT (20%-70% in mice and rats) 124 and AF (60%-90% in mice) 125 . Electrophysiological study of isolated myocytes from acute and chronic infarcted hearts has shown both shortening of the myocardial effective refractory period and slowing of condition time in the left atria, infarct, and border zone. The proposed mechanism for AF induction is reduced expression of Cx40, dephosphorylation of Cx40 and Cx43, and redistribution from the intercalated disc to the lateral cell membrane. Established models for rabbit myocardial ischemia focus extensively on ex vivo studies using Langendorf perfusion systems. After infarction, rabbit myocardium shows prolonged APD and Ca2 + transients ex vivo 126 . Unlike mice and rats, infarction in rabbits leads to delayed AV nodal conduction becaue of fibrosis and reduction in Cx40 expression but did not affect the ventricular effective refractory period 127 . No arrhythmia induction protocol was used in these studies. Dog models focus on atrial ischemia and arrhythmias, with ~ 40% of animals developing AF 128 . Increased INCX current, spontaneous Ca2 + leak, and conduction heterogeneity were found in myocytes from the border zone of the atrial infarct, supporting both triggered and reentry as the mechanism in this model 129 . The use of beta-blocker (nadolol) and Ca2 + channel inhibitor (nifedipine) were more effective at suppressing atrial arrhythmia in this model than class Ic (flecainide) or class III (dofetilide) antiarrhythmic drugs 130 . Pig myocardial ischemia models are well established as they are of similar size and physiology as humans. Ischemia can be induced by either transient intravascular occlusion of the coronary artery or chronic occlusion with an ameroid constructor 131 . Acutely, pigs are exquisitely sensitive to ischemia because of lack of functional collaterals at baseline, which leads to significant procedural mortality from VF 132 . After chronic ischemia, SCD due to spontaneous ventricular arrhythmias occurs in ≈60% to 70% of animals by 3 months 133,134 . Myocytes in the remote zone from have a decreased rapid delayed rectifier K + current (IKr), altered Na + -Ca2 + exchange current (INCX), and increases of late Na + current (INaL), Ca2 + -activated K + current [IK(Ca)], and Ca2 +-activated Cl − current [ICl(Ca)]. In addition, myocytes in the border zone show the same changes along with a decrease of L-type Ca2 + current (ICaL), a decrease of inward rectifier K + current (IK1), and arrhythmogenic SR Ca2 + release-induced EADs and DADs 135 . These changes in the current lead to shortening of the APD in the border zone and prolonged APD in the remote region, setting up a substrate for both triggered and reentrant arrhythmias. Gene therapy with dominant-negative K + channel (KCNH2-G628S) 136 or Cx43 137 reduced VT induction by prolonging the ADP and effective refractory period in the border zone. Limitations: Mouse: Single left coronary artery leads to variation in severity of injury. Model require PES with catecholamine challenge to induced arrhythmias.
Rat: Requires PES and catecolamine challenge to induce ventricular arrhythmias reliably but rare spontaneous arrhythmias.
Rabbit: PES done as ex vivo in Langendroff system. Dog: Atrial ischemia extensive studied. Pig: High mortality due to poor collateral circulation, early spontaneous VT/VF during injury and late model of SCD.
Sheep: Model of heart failure with reduced ejection fraction, study of arrhythmias.

Complete heart block and AV node ablation
Complete heart block leads to AV dyssynchrony and bradycardia, which acutely causes reduced cardiac output and induces volume overload. Compensatory hemodynamic changes occur to improve cardiac function, including ventricular dilation, hypertrophy, and increased stroke volume but are not able to fully restore cardiac output 138 . While genetic models are available for primary complete heart block, secondary complete heart block, which is characterized by fibrosis and necrosis of the AV node, is more difficult to reproduce. Outside histological remodeling, little is known about the molecular changes associated with secondary complete heart block. Given the size, mouse AV node is challenging to identify without immunostaining 139 . Genetic have generated to establish the important transcription factors and ion channels in the AV node (noted in the above section atrioventricular block). Interestingly, disruption of tissue resident macrophages in the AV node, by either knockout of Cx43 in these cells or by genetic ablation of macrophages using diphtheria toxin receptor/diphtheria treatment, lead to progressive AV conduction block 140 . While these resident macrophages were noted in human AV nodes, it unclear if the presence or absence in of these cells contribute to human disease. Electrical needle AV nodal ablation in rats has been described, as it was noted that alcohol injection leads to either transient block or mortality based on the amount used 141,142 . This is a technically challenging model with variable success 142 and high early mortality because of bradycardia and ventricular arrhythmias, as only 6-month-old female rats survived past 3 days 143 . Regardless, the surviving rats recapitulated the cardiac remodeling in humans, with 80% showing spontaneous TdP at baseline which could be induced to sustained VT with PES and isoproterenol challenge 144 . The dog model of AV ablation by transvenous catheter injection for formaldehyde has been well established and leads to compensated hypertrophy and QT prolongation 140,143 . This is due to APD prolongation in the setting of bradycardia, with ≈50% of animals developing spontaneous and 90% drug-induced TdP 144,145 . Chronically, this reduced IKs current and enhanced Ca2 + influx by NCX, leading to increased SR Ca2 + content 142 , which increases the risk for DADs 146 . In contrast, goats undergoing AV ablation did not show APD prolongation but led to increased PLB (phospholamban), Troponin-I and myosin light chain kinase by PKA and RyR2 by CaMKII, suggesting increased Ca2 + sensitivity in this model 147 .

Chronic tachypacing
Overriding the normal conduction system has many deleterious effects but is often reversible. Rapid atrial heart rate or pacing can lead to tachycardia-mediated heart failure, and long-term RV pacing in humans can lead to ventricular dyssynchrony, reduced cardiac output, and heart failure 146 . Long-term pacing has been shown to be detrimental to LV, with significant wall motion abnormalities and perfusion defects in the inferior and apical walls without corresponding coronary disease 148 . Conversely, cardiac resynchronization therapy improves HF outcomes for patients with HF and left-bundle branch block. RV pacing has been used as a model of nonischemic cardiomyopathy, developing significant systolic dysfunction. Long-term RV pacing in AV nodal ablated dogs show the same perfusion mismatches seen in humans and was associated with increased sympathetic innervation of the ventricles 149 . Overdrive RV pacing for 4 weeks in dogs can induce spontaneous ventricular arrhythmias and SCD in 25% of dogs 150 . Myocytes isolated from paced ventricles showed prolonged ADP with reduced Ito currents 151 , likely due in part to downregulation of Kv4.3 152 and increased INa,L 153 in failing heart. In addition, Ca2 + transients showed reduced amplitude, slowed relaxation, and blunted frequency dependence due to reduction in SERCA2a and upregulation of NCX in failing myocytes 154 . Cardiac resynchronization therapy in this model was showed to normalization of APD, reduce the INa,L current, and prevent the negative remodeling associated with heart failure in this model 153,155,156 . VF can be induced in pig by applying AC current to the RV, but using arrhythmic drugs to improve resuscitation was not seen 157 . Ventricular pacing in dogs also leads to secondary atrial fibrosis, dilation, and reduced function as the LV fails, inducing AF 158,159 .
As with rapid ventricular pacing, rapid atrial pacing leads to a reduction in the Ito, in addition to ICa,L and IKs currents 160 . If pacing is stopped and the animal is allowed to recover, the ion currents return to normal, but fibrosis remains, leading to persistent AF. Studying atrial cells from dogs undergoing both rapid atrial and ventricular pacing has shown the atrial ion channel expression in HF, AT, and HF with AT can be significantly different 161 . Upregulation of profibrotic miRNA has also been described in atrial paced dogs, providing a novel target to prevent atrial fibrosis and AF 162,163 . In rabbits, rapid atrial pacing increases atrial fibrosis and TGF-β signaling, which can be attenuated with losartan 162 . AF can be induced in 40% of rabbits in this model. Atrial pacing leads to decrease KCNE1 KCNB2 expression, reduced IKs, and shortening the AERP 164 . This was thought to be due to microRNA-1 upregulation in the atria. In sheep, natriuretic peptide release is found immediately after RV pacing, returning to normal after cessation 165 . In goats, chronic atrial pacing led to atrial dilation, with reduced PKA phosphorylation of PLB and increased CaMKII phosphorylation of RyR2, leading to reduced SR Ca2 + load 147 . While structural changes similar to humans with tachycardia mediated cardiomyopathy are seen, increased arrhythmogenesis has not been reported. Given their small size, in vivo pacing is difficult in mice. Tethered epicardial pacing has been used to study AV dyssynchrony and synchrony in mice after I/R injury. Dyssynchrony leads to further deterioration of cardiac function and activation of p38, ERK1/2, JNK, and MSK1 and inhibition of the GSK3β pathways. This was reversed by resynchrony 166 . Recently, the development of fully implantable epicardial micro pacing technology may allow for longitudinal pacing studies 167 . In rats, initiation of rapid atrial pacing leads to upregulation of multiple voltage-gated K + channels (Kv1.5, Kv4.2, and Kv4.3) 168 , which contribute to repolarization by I Kr and ITo. After 2 days of atrial pacing, AF can be induced in ≈20% of animals, with upregulation of associated AF genes (CASQ2, KCNJ2, and TGFB) and activation of the TGF-β and IL-6 pathways 169 . Rapid transesophageal pacing of the LV has been used to reliably induce VF to study the effects of medication for resuscitation 170

Inflammation
Myocardial inflammation is a known driver of atrial and ventricular arrhythmias 171,172 . Postprocedural arrhythmias are common after cardiothoracic surgery but, while they are usually self-limiting, they lead to prolonged hospital stays 173 . While there is usually a preexisting arrhythmogenic substrate due to the underlying disease, surgical scarring and inflammation further exacerbate the system, leading to arrhythmia. Large cohort studies have shown elevated proinflammatory cytokines associated with persistent AF, including CRP [174][175][176] , TNF-α, IL-1β, IL-6, and IL-10 177,178 . After surgery, there is an increase in both macrophages and neutrophils to the surgical site, with reactive oxygen species production from MPO (myeloperoxidase) activity 179,180 . Inflammation because of myocarditis is more complex, and the course of the acute and chronic phase of the immune response www.nature.com/scientificreports/ is dependent on the underlying cause (infectious versus rheumatological). The cytokine profiles are found in viral and autoimmune myocarditis includes more proinflammatory monocyte infiltration and myocyte necrosis than postsurgical injury 181 . This leads to a multitude of ECG changes, including sinus tachycardia, widened QRS patterns, low voltage, prolonged QT, variable AV blocks, and diffuse ST-elevations [182][183][184] . Sterile inflammation has been used to induce AF in dogs 183 and sheep 185 . Pericardial talc treatment of dog atria to induce sterile inflammation induced AF in ≈60% of dogs and was significantly reduced with topical steroids or NSAIDS 185 . In sheep, treatment with atorvastatin reduced hs-CRP, IL-6, and TNF-α expression, which improved the atrial effective refractory period at 72 hours 186 . While inflammatory myocarditis can be induced in rats 187 and guinea pigs 188 , no current reports on the arrhythmia potential are available. Currently, there are 2 models of viral myocarditis due to exposure to coxsackievirus B3 (CVB3) 189 and encephalomyocarditis virus A (EMCV) 190 . There are several issues with the viral myocarditis models in animals. First, of the viruses primarily associated with human myocarditis (parvovirus B19, herpes simplex 9 and coxsackievirus B3), only CVB3 is infectious to animals, with EMCV only rarely causing human disease newborns. Second, the CVB3 mouse model is highly inflammatory and more mimics childhood infections than the milder course in adults 191 . Third, only particular stains of mice are susceptible to viral infection. Regardless, C3H/He mice exposed to develop similar arrhythmias to humans (80% sinus arrest, 30% second or third-degree AV block, 30% PACs, 20% PVCs, and 10% VT) 192 . Ex vivo electrophysiological studies showed no change to the APD, but mice exposed to CVB3 were hyperpolarized with slightly increased VERP 193 . EMCV exposed DBA/2 mice develop AV block in 40% of mice over 2 weeks, with two-thirds of those mice showing mononuclear cell infiltration and edema and another onethird showing necrosis of the conduction system 194 . No further electrophysiological studies were conducted. Traditionally, myocarditis can be induced in mice by either immunizing with a cardiac structural peptide (myosin heavy chain [MHC-α] or cardiac troponin I) 195 or delivering primed dendritic cells pulsed with MHCα 196 . Importantly, BALB/c mice are susceptible to peptide immunized myocarditis while C57BL/6 strains are resistant, from which a majority of transgenic lines are created 197 . MHC-α peptide-induced myocarditis showed significant immune cell infiltration, increased expression of both TNFα and INFγ, fibrosis and prolongation for the APD in ventricular myocytes, which all could be attenuated with atorvastatin 187,188,198 . Ctla4 + / − Pdcd1 − / − mice spontaneously develop myocarditis, modeling immune checkpoint inhibitor-induced myocarditis 199 . These mice succumb to progressive ventricular hypertrophy and SCD early. Progressive AV block and sinus arrest occurs in ~ 30% of transgenic mice, similar to arrhythmia seen in patient with immune checkpoint inhibitor-induced myocarditis.
Further study using this model would greatly improve treatment for patient with adverse events after immune checkpoint inhibitor therapy. Limitations: Mouse: Strain-specific susceptibility. C3H/He and DBA/2 mice susceptible to viral myocarditits while C57BL/6 are protected. BALB/c susceptible to immunogen induced myocarditis while C57BL/6 more resistive.
Rat/Guinea Pig/Dog/Sheep: Model of AF, but nonphysiological induction of inflammation with talc.

Metabolic-and drug-induced arrhythmia
Dietary and metabolic considerations also contribute to atrial arrhythmia development as there is a known association with BMI and diabetes in AF 200,201 , and incidence of paroxysmal AF is reduced after gastric bypass surgery 202 . Off-target drug effects lead to adverse clinical outcomes, particularly arrhythmias induction due to block of delayed rectifier K + channels, IKr, causing drug-induced long QT syndrome and TdP ventricular arrhythmias. Streptozotocin-induced diabetic models have been developed for both mice and rats 203 and consistently shown prolonged APD, increased sympathetic innervations, and inflammation. Studies have shown this is likely due to production of advanced glycation end products (AGEs) in diabetes animals, but they are inconstant in the electrophysiological mechanisms 204 . Initial studies showed a reduction in the Ito current as the underlying cause of AF [205][206][207] , while others suggest changes in sinoatrial node connexin channel expression 208,209 , atrial myocyte Ca2 + handling proteins (TRPC1/6, RyR3) 210 and AV nodal ion channels (TRPC1, CASQ2, RYR2, and RYR3) 211 are involved. Diabetic mice and rats also have reduced K + channel expression, leading to overall reduced K + currents, which was dependent on glycosylation of CaMKII and activation PKC 212 . Further studies showed in the setting of hyperglycemia, mice had more diastolic calcium leak through RyR2, prolonged ADP90, ADP alternans, increased DADs, and frequent premature ventricular complex, which could be suppressed by genetic inhibition of CaMKII 213 . Blocking IL-1β activation of CaMKII in this model restores the APD to normal and suppresses VT induction in explanted hearts from diabetic mice 214 . These studies all provide a central role for CaMKII in regulating ion channel expression and function in hyperglycemia. Diet-induced obesity has been shown to affect inflammation and gene expression in multiple disease models. Mice fed 3 months of a high-fat diet had a 15% increase in their QTc and increased IKs current than mice on a normal diet and developed a tenfold increase in premature ventricular complex burden 215 . The increased IKs current was thought to be due to the reduced expression of voltagegated K + channels. Diet-induced obesity mice were also found to have reduced NaV1.5 expression and current, leading to reduced ADP and conduction velocity in the atrial and increased incidence of induced AF 216 . Diet-induced obesity rats over 8 weeks have increased expression of CaV1.2, HCN4, Kir2.1, RYR2, NCX, and SERCA2a in the LV, which may contribute to DADs and triggered PVCs 217 . Rabbits fed a high-fat diet had increased cardiac sympathetic innervation (as seen by increased GAP23 expression), prolonged ADP and increased ICa, which led to QTc prolongation and repolarization heterogeneity in the ventricle 218 . Isolated hearts were more susceptible to VT induction. While these studies highlight the important changes to ion channel expression animals due to a high-fat Western diet, further study is needed to linking these findings to humans. A specific line of inbred rats (Fischer F344 at 20-24 months) develop age-dependent adverse cardiac remodeling, with males developing more cardiomyocyte hypertrophy, intestinal fibrosis, and systolic dysfunction and females with more cardiac hypertrophy and diastolic dysfunction 219  www.nature.com/scientificreports/ show enlarged atrial, fibrosis, and CD68 + monocyte infiltration, similar to human disease221.452 Both male and female Fischer 344 rats are more susceptible to AF induction by atrial pacing, with 80% of animals showing atrial arrhythmia 220,221 . These are the only models of spontaneous AF in aged animals and could provide important insight to mechanism and future therapies for AF in our aging population. In rabbits treated with clofilium (K + channel blocker), 70% of animals develop TdP, which can be increased to 100% with the addition of α1 agonist methoxamine 222 . Blockade of CaMK (calmodulin kinase) or PKA in this model reduced pausedependent VT that was independent of these kinases effect on L-type Ca2 + channel activity 223 . Interestingly, the dose-dependent CaMKII blockade did not change the QT interval, unlike the dosedependent PKA blockade. Further study showed that blocking CaMKII reduced the ratio of the TU interval, which is likely more critical than the QT interval in the induction of pause-dependent VT 224 . Limitations: Mouse: Streptozotocin and DIO models well established, increased susceptibility to AF and VT with PES. Rat: Age-dependent fibrosis found in Fisher 344 rat strain, model of AF. Rabbit: Established model of clofilium-induced TdP.

In vivo approaches
The following are examples of in vivo models used to evaluate potential new antiarrhythmic drugs:

Arrhythmia of chemical origin
Arrhythmias can be caused by many drugs alone or in combination. Arrhythmias result from the concomitant use of sensitizing drugs such as intravenous adrenaline and anesthetics such as ether, chloroform, or halothane. These arrhythmic substances have different effects in different animal species.

Limitations-comments:
Male Ivanovas rats: Rats with Aconitine Antagonism: With regard to ventricular extrasystoles, tachycardia, fibrillation, and death, the antiarrhythmic action of the test substance is quantified.
Guinea Pig: Arrhythmias in Whole Guinea Pigs Caused by Aconitine: induce ventricular arrhythmias in a whole animal model.
Male Marioth guinea pigs: Digioxin developed arrhythmia in guinea pigs: ventricular premature beats, fibrillation, and cardiac arrest.
Dogs: Adrenaline-induced Arrhythmia: induce ventricular arrhythmias in a whole animal model. Rats: Calcium-induced arrhythmia: ventricular flutter and fibrillation.

Rats with aconitine antagonism
Activating sodium channels over time, aconitine, a plant alkaloid from aconitine root, causes ventricular arrhythmias. Rats that have consumed aconitine can be used to test drugs that are thought to have anti-arrhythmic effects. Urethane (1.25 g/kg) is administered intraperitoneally to anaesthetize male Ivanovas rats (300-400 g). Aconitine (5 ug/kg) is dissolved in 0.1 N HNO 3 and administered 0.1 ml/min continuously into the rat's saphenous vein. Every 30 s, an ECG in lead II is recorded. 5 min before to the infusion of aconitine, a test substance is administered intravenously or orally. The test group receives a larger dose of aconitine than the untreated group, which results in a measure of antiarrhythmic activity. With regard to ventricular extrasystoles, tachycardia, fibrillation, and death, the antiarrhythmic action of the test substance is quantified by the amount of aconitine/100 g animal (infusion duration) 225,226 .
Arrhythmias in whole guinea pigs caused by aconitine Sodium pentobarbital (50 mg/kg i.p.) was used to anesthetize guinea pigs weighing between 300 and 400 g. In order to keep pCO2, pO2 and pH in the normal range, respiration was maintained by artificial respiration (ambient air, volume 1.5 ml/100 g, frequency 55 beats per minute).Aconitine and study drugs were administered through a polyethylene tube inserted into the right jugular vein. A cannula was inserted into the left carotid artery to measure systemic blood pressure. The ECG leads of the articulated limbs were recorded. A polygraph recorder (NEC San-ei Instruments Ltd., Tokyo, Japan) was used to continuously monitor the subjects' blood pressure, heart rate and ECG (Leads I and II). Vehicle or NCX inhibitors were administered to different groups for each drug or vehicle dose (ie, 0-30 mg/kg) by intravenous bolus injection in the jugular vein after 15 min of stabilization.
To induce ventricular arrhythmias in a whole animal model (Lu and Clerck, 1993) ( Fig. 1, Protocol I), 25 g/kg aconitine was administered five minutes later.Each animal received a single dose (treatment) of vehicle or one of the NCX inhibitors. The doses of SEA and KBR were 1 to 10 mg/kg and 1 to 30 mg/kg, respectively (Fig. 7) 227 .

Digioxin developed arrhythmia in guinea pigs
Overdose with digoxin leads to ventricular premature beats, fibrillation and death. Antiarrhythmic drugs prolong the duration of these symptoms. Male Marioth guinea pigs (350-500 g) are anesthetized by intraperitoneal administration of sodium pentobarbital (35 mg/kg). The animal is catheterized under mechanical ventilation (45 breaths per minute) in the trachea, jugular vein and carotid artery. Digoxin is administered into the jugular vein using an infusion pump at a dose of 85,g/kg.266 mL/min to cardiac arrest. Steel needle electrodes are used to record the ECG throughout the experiment. The carotid artery is used to measure blood pressure. The study drug will be administered intravenously or orally one hour before the infusion. Note the interval between ventricular premature beats, fibrillation, and cardiac arrest. The total amount of digoxin delivered (μg/kg) to www.nature.com/scientificreports/ induce VF, VF and cardiac arrest after treatment with study drug is statistically compared to a control group receiving digoxin alone 228 .

Strophanthin/ouabain-induced arrhythmia
The carotid artery is used to measure blood pressure. The study drug will be administered intravenously or orally one hour before the infusion. Note the interval between ventricular premature beats, fibrillation, and cardiac arrest. The total amount of digoxin delivered (μg/kg) to induce VF, VF and cardiac arrest after treatment with study drug is statistically compared to a control group receiving digoxin alone. The test material is administered 10 min after the arrhythmia has resolved. The test substance is considered antiarrhythmic if the extrasystoles cease immediately after ingestion of the drug. If the study drug does not show a positive effect, higher doses are given 15 min apart. If the test chemical is effective in reversing the arrhythmias, the next dose is administered when a sustained arrhythmia recurs 229,230 .

Adrenaline-induced arrhythmia
High doses of adrenaline may cause arrhythmia to occur. Pentobarbitone sodium (30-40 mg/kg) is administered intraperitoneally to dogs (10-11 kg) to induce anaesthesia. A cannula is placed in the femoral vein, and 2-2.5 g/ kg of adrenaline is then infused. Atrial and Lead II ECGs are both recorded. Three minutes after the infusion of adrenaline, the test medication is given 231 . A test substance is deemed to have an antiarrhythmic action if extrasystoles stop occurring right away after taking the medication.  www.nature.com/scientificreports/ Calcium-induced arrhythmia Nembutal (60 mg/kg) is administered intraperitoneally to anesthetize albino Wistar rats (60-130 g). 2 ml/kg of a 10% aqueous solution of calcium chloride is injected into the femoral vein to induce ventricular flutter and fibrillation. Using a cardioscope connected to the animal via two percutaneous precordial clamp electrodes, the rhythm and behavior of the heart are observed during and two minutes after the injection.A test drug is administered two minutes before the calcium chloride infusion 232 .
The study, Tinospora cordifolia (T. cordifolia) alcohol extract was tested for its ability to prevent Cacl 2 -induced arrhythmia. Rats were given intravenous infusions of Cacl 2 (25 mg/kg) to induce arrhythmia. Then, T. cordifolia extract (150,250, and 450 mg/kg) and verapamil (5 mg/kg, iv) were administered to the animals. Electrocardiogram in Lead II was seen. We measured the levels of potassium, sodium, and calcium in the plasma. Verapamil decreased the heart rate by 9.70%, T. cordifolia at 150, 300, and 450 mg/kg by 26.30%, 29.16%, and 38.29%, respectively, while Cacl 2 decreased the heart rate by 41.10% when compared to the control group. In rats given verapamil and T. cordifolia, the PQRST waves were restored to normal, and atrial and ventricular fibrillation were controlled. Cacl 2 raised the levels of calcium and salt while lowering potassium in the blood. T. cordifolia

Ventricular fibrillation electrical threshold
When comparing antifibrillators, consider the maximum rate at which the atria can respond to stimulation. Ventricular thresholds were assessed using a variety of electrical pacing techniques, including sequential pulse pacing, continuous 50 Hz pacing, and single pulse pacing. After intraperitoneal administration of pentobarbital sodium (35 mg/kg) to anesthetize dogs (8-12 kg), they are kept alive by artificial respiration. 's chest is opened, the heart is suspended over the pericardium, and blood pressure and temperature measurements are taken. The SA node is compressed and a Teflon disk sutured to the anterior surface of the left ventricle is implanted with the Ag-AgCl pacing electrode 234 . The pilot electrode is supplied with a constant anode current for 400 ms (3 ms squared). A computerized stimulator is used to program the electrical stimulation. A recording electrode is mounted outside each chamber. The ventricular fibrillation threshold (VFT) is determined by giving a value between 0.2 and 1.8-s train of 100 ms 50 Hz pulses for every 18 primary motor stimuli. VFT is the minimum pulse train current required to produce sustained VF. The heart is rapidly defibrillated at the onset of ventricular fibrillation and then has 15 to 20 min to return to a controlled state. The femoral vein is used to administer medication. VFT is assessed both before and after study drug administration and results are compared using Student's t-test 235 .
Programmed electrical stimulation induced arrhythmia Dogs (8-12 kg) received 30 mg/kg sodium pentobarbital intravenously prior to anesthesia by mechanical ventilation. The cannula is inserted into the external jugular vein. A thoracotomy is performed between the fourth and fifth ribs to expose the heart.The left anterior descending coronary artery (ADL) is the only one. A 20 gauge hypodermic needle is placed over the LAD and a ligature is placed around the artery and needle. Withdrawing the needle creates a large stenosis in the vessel. The LAD infusion takes five minutes. Using a silicone rubber ring, the LAD is blocked for two hours to damage ischemic heart tissue. The critical stenosis is then maintained while the vessel is reperfused for an additional two hours. During the LAD reperfusion phase, an epicardial bipolar electrode is sutured to the interventricul septum, close to the region of obstruction. Silver disc electrodes that are subcutaneously implanted are used to track the ECG. The chest is reopened after 6-9 days, and planned electrical stimulation is carried out using an electrode implanted in a noninfarcted location. After 15 pacing www.nature.com/scientificreports/ stimuli, a 200 ms pacing stimulus is given as a final stimulus. Animals with persistent ventricular tachycardia and ventricular fibrillation are used in the investigation. Heart rate and ECG intervals are measured prior to the start of the planned electrical stimulation. The critical stenosis is then maintained while the vessel is reperfused for an additional two hours. During the LAD reperfusion phase, an epicardial bipolar electrode is sutured to the interventricular septum near the site of obstruction. Silver disc electrodes implanted under the skin are used for ECG monitoring. The chest is reopened after 6 to 9 days and elective electrical stimulation is given with an electrode implanted at a non-infarcted site. After 15 pacing stimuli, a stimulus lasting 200 ms is delivered as the last stimulus.Animals with sustained ventricular tachycardia and ventricular fibrillation are used for the study. Heart rate and ECG intervals are measured before programmed electrical stimulation begins. The study drug will then be administered after 30 min. The minimum current required to induce sustained VF is recorded before and after study drug administration, and the mean results of 10 experiments are compared using Student's t-test 234 .

Dog model of sudden coronary death
Sudden coronary death is one of the leading causes of death in industrialized countries. This dog model is used to investigate protection against sudden cardiac death. Male mongrel dogs weighing 14 to 22 kg are given intravenous pentobarital sodium (30 mg/kg) for sedation. The cannulas are inserted into the animal's trachea and maintained by artificial respiration. A cannula is inserted into the jugular vein to inject the test substance/ saline solution.Once the chest is opened to show the heart, the left anterior descending coronary artery (LAD) is isolated and a 20G needle placed over it. Once the ligature is tied across the artery and a key stenosis develops, the needle is withdrawn. For severe strictures, the LAD is closed with a silicone rubber ring for two hours, followed by reperfusion for an additional two hours. Bipolar stainless steel epicardial electrodes and bipolar dip electrodes are sutured to the left atrial appendage and interventricular septum, respectively, to stimulate the left atrium. Two equivalent stainless steel electrodes are sutured to the wall of the left ventricle, one at the LAD position distal to the block and the other at the position of the left peripheral coronary artery (LCX). A silver-coated electrode is placed adjacent to the heart surface and penetrates through the wall into the LCX lumen. The silver disc electrodes for ECG monitoring are inserted via implants under the skin. The surgical incision is then closed and the animal is allowed to heal. After recovery, the animals receive the study drug. A direct anode current of 15 A from a 9-V nickel-cadmium battery connected to a subcutaneously implanted puck electrode is used to record a lead ECG II for 30 s every 15 min on a cardiac cassette recorder. If ventricular fibrillation or constant anodic current occurred for 24 h, the animals were sacrificed. After the hearts are removed, the thrombus mass in the LCX is removed and weighed. The heart is divided into slices and stained with tetrazolium triphenyl chloride (TTC stain) to determine where the infarction occurred. Investigations into the onset of lethal arrhythmia and ventricular ectopy are conducted using recordings from the cardiocassette evaluation of tachyarrhythmias, both sustained and not 235 .

Exercise-related ventricular fibrillation
Coronary artery stenosis can be used in exercise-based studies that better mimic the circumstances of patients with coronary artery disease. This model can be used to assess how antiarrhythmic drugs affect various cardiovascular parameters during exercise and ischemia testing. Hybrid hearts weighing 15-19 kg are exposed after anesthesia with sodium pentobarbital (10 mg/kg, intravenously) and supported by the pericardial mount. The left circumflex artery is surrounded by a hydraulic obturator and a 20 MHz pulsed Doppler flow transducer. A pair of silver-plated insulated wires was used to suture the epicardial surfaces of the left and right ventricular electrograms, and a Gould biotachymeter was used to measure heart rate. In the next step, the left ventricle is punctured with a solid-state pressure transducer that has already been calibrated. The left anterior descending coronary artery is then partially occluded for 20 min before ligating. Leads from the cardiovascular system were inserted under the skin and tied around the animal's neck. To relieve the pain, the animals are given antibiotics and painkillers. Three to four weeks after the onset of myocardial ischemia, animals learned to sit still on a laboratory bench. Then run on a motorized treadmill during this healing period. VF sensitivity is then assessed on a motorized treadmill. The animals walked at 6.4 km/h (0% incline) for the first three minutes of treatment. The odds increase by 0%, 4%, 8%, 12% and 16% every three minutes. In the last minute of exercise, the treadmill is stopped, the left circumflex artery occluded, and the blockage maintained for an additional minute (total occlusion time 2 min). If the animal loses consciousness, an electrical defibrillator is used. In the case of ventricular fibrillation, the obstruction is released. The stress plus ischemia test is performed after pre-treatment with the test drug and the results are compared to those of the control group (saline).The effective refractory period is calculated the following day using the Medtronic Model 5325 programmable pacemaker, both at rest and during myocardial ischemia. In addition, the effect of the test chemical on coronary blood flow is measured using a flow meter 236 . Rate of change of left ventricular pressure and other hemodynamic data are recorded with a Gould Model 2800 S eight-channel recorder. Using analysis of variance, the response of reactive hyperemia to each refractory block is assessed after averaging the results. To investigate how a pharmacological intervention affects the occurrence of arrhythmias, the chi-square test with Yate's correction was performed. Reperfusion arrhythmia in rats Ventricular arrhythmia and myocardial infarction are the outcomes of ligation of the left major coronary artery. In the course of the ligation and subsequent reperfusion, an electrocardiogram is taken. In cardiac slices, p-nitro blue tetrazolium chloride staining is used to quantify the amount of infarcted tissue. Pentobarbitone sodium (60 mg/kg) is administered intraperitoneally to anaesthetize Sprague-Dawley(350-400gm) rats. The jugular vein is cannulated to provide test medications while the animal is kept on artificial breathing. Using a pressure transducer attached to a polygraph, blood pressure is taken from the carotid artery. The heart is visible when the chest is opened. In the case of infarct size investigations, the left coronary artery is found, ligated for 15 to 90 min, and then re-perfused for 30 min. Five minutes prior to the ligation, test medication is given. Throughout the entire experiment, peripheral blood pressure and ECG lead II are continually recorded. During the occlusion and reperfusion periods, the number of premature ventricular beats, ventricular tachycardia, and fibrillation are counted 237,238 . The animal is sacrificed at the conclusion of the reperfusion phase, and the infarct size is measured using TTC (p-nitro blue tetrazolium trichloride) staining. To visualise the infarct tissue (blue/violet, stained healthy tissue, unstrained necrotic tissue) the heart is dissected, sliced into transverse Sects. (1 mm thick), and stained with TTC produced in Sorensen phosphate buffer containing 100 mM, L-maleate. Slices are imaged, and the baby area is calculated using planimetry using all of the slice projections. Comparing drug-treated animalsvariation in hemodynamic parameters and infarct size to control values.

Reperfusion arrhythmia in dogs
Dogs with coronary artery ligation may experience elevated blood pressure, heart rate, heart contractility, left ventricular end diastolic pressure, and ventricular arrhythmias, particularly during the reperfusion period. The anaesthetic thio-butobarbital sodium (30 mg/kg intraperitoneally) is administered to dogs (20-25 kg) to induce anaesthesia. The dogs are then maintained on intravenous chloralose (20 mg/kg) and 250 mg/kg urethane intravenously, followed by the subcutaneous delivery of 2 mg/kg(morphine). Animal is then kept on an artificial breathing system: For the purpose of administering the test substance, the saphenous vein is cannulated, and lead II of the ECG is constantly monitored. A pressure transducer is attached to a cannulated femoral artery to measure blood pressure. The left ventricular pressure curves are used to calculate heart rate and left ventricular end diastolic pressure. An increase in left ventricular pressure is used to evaluate myocardial contractility. Rats were used in the experiment in a similar manner. 90 min are spent ligating the coronary artery. The test substance is given twenty minutes before ligation. The animal is reperfused for 30min 239,240 . During the course of the experiment, all of the mentioned parameters are recorded.
Comparing drug-treated animals to vehicle controls reveals changes in metrics (mortality, hemodynamics, and arrhythmia).

Two stage coronary ligation in dogs
Methohexitone sodium (10 mg/kg) is administered intravenously to anaesthetize dogs (8-11 kg) and keep them breathing artificially. The heart is visible when the chest is opened. Two phases of coronary ligation are carried out after the location of the left coronary artery. A 21-gauge needle and two ligatures are put around the artery. The needle is removed after the initial ligature has been tied around the artery. The second ligature is firmly secured around the artery 30 min later. After the second ligature has been knotted for 30 min, the chest is closed in layers and the dog is given time to recover. Arrhythmias start to appear after 24 and 48 h following ligation and disappear after 3-5 days. The atrial electrogram, lead II ECG, and mean blood pressure are all recorded. Following coronary artery ligation, test medications are administered as an infusion for 10 minutes 241,242 . Animals given drugs are compared to vehicle controls for changes in metrics (mortality), hemodynamics, and arrhythmia).Boyden and Hoffman's canine model, which causes tricuspid regurgitation and right atrial enlargement by banding the pulmonary artery, may also have a clinical analogue in people who have both tricuspid regurgitation and chronic obstructive pulmonary disease 243 . Instead of an anatomical barrier, a functional zone of blocking and area of sluggish conduction create the conditions for re-entry in these dogs. The first documented model of canine atrial flutter using sterile pericarditis also shows functional entrance 244,245 . Atrial flutter frequently occurs in patients after cardiac surgery, and this may be associated to postoperative sterile pericarditis, which is why this model was created.

Genetically induced arrhythmia
It has been reported that a group of German shepherd dogs have inherited ventricular arrhythmias and are more prone to abrupt demise, which typically happens while they are sleeping or at rest following an active or exciting day. These canines can be used to evaluate possible antiarrhythmic medications. Although there is commonly noticeable T wave notching on the ECG, the QT interval is not prolonged. The ventricular tachycardias are quick polymorphic, follow lengthy R-R intervals, and are almost certainly brought on by early depolarizations of the Purkinje system, which activate the triggered activity. Both the transient outward current's density and its time constant of inactivation are decreased in the epicardial myocytes 246 . Additionally, there are deficiencies in cardiac sympathetic denervations. This dog model initially reminds one of the congenital long QT-syndrome, which is characterised by bradycardia-induced polymorphic ventricular tachycardia, rapid mortality, and genetic abnormalities in ion channels that control repolarization. The dogs do not, however, exhibit a prolonged QT www.nature.com/scientificreports/  Tables 2 and 3 list published animal models of genetic arrhythmia syndromes. Most animal arrhythmia models are mice, given the ease of genetic manipulation. Despite differences between rodent and human cardiac electrophysiology (Fig. 2), mouse models have enabled the study of human genetic diseases, identification of pathogenic mutations, characterization of disease pathophysiology, and testing/screening of therapeutic interventions. Advances in gene editing technology such as CRISPR/Cas9 have provided faster, easier, and cheaper methods to develop genetic models in animals and cells. Compared with cellular models such as human-induced pluripotent stem cells, animal models provide a distinct advantage in modeling cardiac arrhythmias where the anatomy of the heart is relevant 247 . In the following section, each of the major genetic arrhythmia syndromes are introduced, followed by examples of animal models that have informed disease pathophysiology and treatment approaches.

Channelopathies
Channelopathies are caused by mutations in ion channel genes or genes that regulate ion channels and generate arrhythmia risk in the structurally normal heart. However, overlap syndromes caused by mutations in channelopathy genes (eg, SCN5A) can also be associated with alterations in cardiac structure, which are discussed in Section genetic arrhythmia syndromes associated with structural heart disease. Catecholaminergic Polymorphic Ventricular Tachycardia CPVT is characterized by arrhythmogenesis evoked by elevated catecholamines during stress or exercise. CPVT is caused by gain-of-function mutations in proteins that constitute the intracellular Ca2 + release unit of the SR. These mutations result in spontaneous Ca2 + release from RYR2 SR Ca2 + release channels, raising diastolic Ca2 + and generating membrane depolarizations and delayed afterdepolarizations via Ca2 + extrusion through the electrogenic Na +-Ca2 + exchanger. Pathological mutations in RYR2 make up more than half of identified CPVT cases and are inherited in an autosomal dominant fashion. Mutations in regulatory partners of RyR2 are rarer and include calsequestrin, calmodulin, and triadin. Evidence suggests that KCNJ2 mutations may also be involved in CPVT. Mice have been the primary animal model for studying CPVT and have been valuable not only to establish arrhythmia pathophysiology but also to identify new drug therapy that proved efficacious in humans 248 . At least 1 attempt to generate a rabbit model overexpressing human RYR2-R4497C was unsuccessful, likely due to the selected promotor and size of the transgene 249 . The first animal model of CPVT generated was an RYR2-R4496C ± knock-in mouse 29 , which had exercise-and catecholamine-inducible ventricular arrhythmias. Since then, several mouse models have been designed that carry RYR2 mutations identified from patients with CPVT (see Table 1). When loss-of-function mutations are combined with gain-of-function mutations, mice are protected from CPVT 250 . Identifying the pathogenicity of specific mutations is important because RYR2 mutations are implicated in several other arrhythmia disorders, as detailed below. Interestingly,  www.nature.com/scientificreports/ a RYR2-exon3 deletion was identified in patients with a severe form of CPVT, but the corresponding mouse model failed to reproduce the CPVT phenotype 251 . Cardiac calsequestrin (CASQ2) mutations are inherited in an autosomal recessive manner, leading to loss of function. Casq2 serves as a high-capacity Ca2 + buffer in the SR and regulates RYR2 gating. In the mouse, CASQ2 deletion causes a severe exercise-and/or catecholamineinduced arrhythmia phenotype consistent with patients lacking Casq2 expression. Observations from CASQ2 mouse models carrying mutations identified from patients show these commonly lead to loss of Casq2 expression in the heart. Recent evidence, however, suggests that Casq2 mutations could also be inherited in an autosomal dominant manner 252 . A knock-in mouse model expressing Casq2-K180R ± reported catecholamine-induced arrhythmias validating this inheritance pattern 253 . Triadin is another protein in the SR Ca2 + release unit where autosomal recessive inheritance has been reported in patients with CPVT. When triadin was knocked out in mice, they developed substantial ultrastructural changes in the junctional SR and reduced expression of proteins comprising the Ca2 + release unit, leading to catecholamine-inducible arrhythmias 254 . Calmodulin genes (CALM1/2/3) encode 3 identical proteins (CaM) and some mutations are associated with CPVT 255 . Zebrafish models have been generated to examine the pathogenesis of overexpressing CALM mutations, and they have successfully demonstrated cardiac arrhythmias (Table 2). Finally, KCNJ2 loss of function mutations, which commonly have been linked to the Anderson-Tawil Syndrome and cause reduced IK1 current (see section Long QT syndrome), have also been identified in patients with CPVT. A knock-in mouse carrying KCNJ2 − R67Q ± had significant evoked arrhythmias without any QT prolongation ( Table 2). The CASQ2 knockout (KO) mouse is an excellent model to investigate CPVT. It has a severe and highly penetrant phenotype, something not always observed with RYR2 mouse models of CPVT ( Table 2). The age of onset for CPVT is only a few weeks old and mice have spontaneous arrhythmias under normal housing conditions 256 . CASQ2 KO mice were used to establish proof of principle for the efficacy of gene-therapy in CPVT 257 . In what may be the only example of its kind for an arrhythmia syndrome, the CASQ2 KO mouse was used to establish the therapeutic efficacy of an existing FDA-approved drug, flecainide, in CPVT 248 . CASQ2 KO mice were instrumental to demonstrate that in vivo, RYR2 block is the principal mechanism of flecainide's antiarrhythmic action 258 . Since its discovery in CASQ2 KO mice, flecainide has become the standard of care for preventing arrhythmias in CPVT patients when betablockers are insufficient 43,259 . CASQ2 KO mice have also recently been used to establish a novel tissue mechanism responsible for ventricular ectopy in CPVT (Fig. 3). To determine the cellular origin of ventricular arrhythmias in CPVT, Blackwell et al. 260 , used conditional murine models with Casq2 expression only in ventricular myocardium or in the specialized conduction system, utilizing the contactin-2 promoter to drive Cre expression and control tissue-specific Casq2 expression. CPVT occurred when Casq2 was deleted in the ventricular myocardium but still expressed in the conduction system. Moreover, catecholamine challenge did not elicit any arrhythmias when Casq2 was deleted in the conduction system but still expressed in the ventricular myocardium. Additional experiments determined that the subendocardial ventricular myocardium juxtaposed to Purkinje fibers is the only cellular source for focal ventricular arrhythmias in CPVT. To understand why that was the case, in silico modeling demonstrated an intriguing phenomenon whereby subthreshold DADs in ventricular myocardium elicit full-blown APs in the conduction system to generate arrhythmias, identifying the Purkinje-myocardial junction as the tissue origin of ventricular ectopy in CPVT (Fig. 3). This discovery could shape treatment and may be critical to our understanding of arrhythmogenesis in other ventricular arrhythmia syndromes where DADs are the cellular arrhythmia mechanism.
Calcium release deficiency syndrome Whereas RYR2 gain-of-function mutations have been associated with CPVT, arrhythmogenic cardiomyopathies, and AF, loss-of-function mutations in RYR2 can cause an arrhythmia syndrome recently termed Ca2 + release deficiency syndrome (CRDS) 261 . In patients with CRDS, exercise stress tests do not provoke arrhythmias. Consequently, this syndrome can escape clinical diagnosis and often presents as sudden cardiac death. In CRDS, arrhythmias are thought to develop because of electrophysiological remodeling. Given the substantial number of benign RYR2 mutations observed, predicting pathogenicity without in vitro or in vivo examination is challenging. CRDS has only been recently described, but 1 knock-in mouse model was generated carrying the patientspecific RYR2-D4646A ± mutant allele 261 . Exercise and catecholamine challenge did not invoke arrhythmias, as predicted. Importantly, this animal model enabled the authors to establish a burst pacing protocol that induced arrhythmias, supporting a possible new clinical diagnostic tool, which was recently confirmed in a clinical study. A previously reported loss-of-function mutation, RYR2-A4860G ± , was identified in a patient with idiopathic VF and subsequently knocked-in to a mouse, but arrhythmias were not reported in vivo 262 . Ex vivo hearts did develop VF in response to isoproterenol but had no arrhythmias in the presence of the RyR2 agonist caffeine. These data hint that this RYR2 loss-of-function mutation may be part of the calcium release deficiency syndrome. Prior work on RYR2 has focused on gain-offunction mutations. CRDS is a relatively new syndrome and animal models provide an opportunity to advance our understanding of CRDS pathophysiology and determine the impact of loss-of-function mutations.

Long QT syndrome
Congenital LQTS often presents as a multiorgan syndrome caused by mutations in proteins responsible for the repolarization of the heart and can cause seizures, syncope, arrhythmia, and sudden death. Alterations in repolarization manifest as prolongation of the AP and, consequently, the QT interval, predisposing the heart to EAD, DAD, and reentrant circuits. Ca2 + , Na + , and many K + channels play a role in cardiac repolarization; accordingly, the genes involved in this syndrome vary and LQTS can be inherited in autosomal dominant or recessive forms. LQTS is traditionally described as a distinct disease manifestation; however, long QT intervals are observed in many overlap syndromes (see Table 2). Approximately 80% of LQTS cases are caused by mutations www.nature.com/scientificreports/ in KCNQ1 or KCNH2, with SCN5A constituting 7% to 10% of cases 263 . It should be reiterated that the currents responsible for mouse and human repolarization are quite different (Fig. 2), and mice are inadequate to model human disease involving mutations in delayed rectifier K + channels (eg, LQT1, LQT2). An aspect of understanding LQT pathogenesis is the observed sex differences in patients, such as QT interval, the onset of cardiac events, and sudden death 264,265 . Animal models allow for significant insight into these investigations since most in vitro models fail to capture the hormonal factors that influence development and regulation. It is noteworthy that sexual dimorphism in QT is not captured by mouse models and may require other animal models such as rabbits 266,267 . Estradiol treatment in ovariectomized rabbits led to prolongation of the QT interval and changes in proteins responsible for repolarization, whereas dihydrotestosterone did not 268 . Another study in rabbits recapitulated findings in patients showing that QT interval was more prolonged in females following treatment with erythromycin 269 .
LQT1 is caused by loss-of-function mutations in KCNQ1 (Kv7.1). Knockout of KCNQ1 in mice leads to characteristics of Jervell and Lange-Nielsen syndrome, causing deafness and prolonged QT interval 270 . Other models have examined distinct mutations with varying phenotypes; however, caution should be used when reviewing the mouse LQT1 literature. Rabbits are a much better model for examining human-like mechanisms of altered repolarization. However, only 1 transgenic rabbit model is reported, which carries cardiac-specific overexpression of KCNQ1-Y315S, leading to LQT and inducible EADs and VT with sympathetic stimulation 271 .
LQT2 is caused by loss-of-function mutations in KCNH2 (Kv 11.1). In contrast to humans, KCNH2 contributes little to repolarization in mice (Fig. 2). As such, mouse models are not realistic for modeling LQT2 and should not be used. A rabbit transgenic model was generated with cardiac-specific overexpression of KCNH2-G628S and developed LQT, spontaneous PVT, and sudden death. Arguably, the most interesting LQT2 model is the work in zebrafish 272 . In 1 study, the endogenous ortholog of hERG (zERG) was knocked down using morpholinos and then various hERG mutants were expressed in its place. The phenotype data (prolonged APD and/or 2:1 AV block) correctly identified 39/39 pathogenic mutants and 9/10 nonpathogenic polymorphisms. Further work using this model could establish the pathogenicity of other variants of uncertain significance.
LQT3 is caused by gain-of-function mutations in SCN5A (Nav1.5). Several mouse models have been generated that recapitulate the LQT phenotype. Both the SCN5A-ΔKPQ/ + and SCN5A-ΔQKP/ + mouse models report many characteristic phenotypes such as prolonged QT interval, a more pronounced T wave, prolonged APD, arrhythmias, and sudden death, which make them suitable for examining LQT3 mechanisms and pathogenesis 273,274 . A successful bench-to-bedside study, conducted in ventricular myocytes isolated from guinea pigs, demonstrated that mexiletine restored the APD in cells treated with the Na + channel inactivation inhibitor, anthopleurin 275 . Mexiletine is now used routinely in the treatment of patients with LQT3. In rare cases, patients have been identified linking LQTS with mutations in the beta accessory proteins for SCN5A. An SCN1B knockout mouse was generated and had long QT, bradycardia, and delayed repolarization; interestingly, it was found that these mice had increased Na + channel expression. Nav1.5 channel gating was unaffected, but peak and persistent currents were increased in isolated cardiomyocytes.
Andersen-Tawil syndrome is a rare LQTS associated with physical abnormalities and hypokalemic periodic paralysis and is primarily caused by loss of function mutations in KCNJ2 (Kir2.1), resulting in reduced IK1 current. Neonatal (1 day old) KCNJ2 knockout mice were characterized by long QT and bradycardia, before dying from complete cleft palate and inability to feed 276 . A more useful arrhythmia phenotype-LQT and spontaneous VT-was observed in the KCNJ2-T75R cardiacspecific overexpression mouse model 277 . The remaining LQTS types result from mutations in K + channels, Ca2 + channels, and key regulators of ion channel function ( Table 2). The ankyrin-B syndrome is characterized as an overlap syndrome, with long QT, sinus node dysfunction, conduction abnormalities, exerciseinduced arrhythmia, VF, and VT. The ankyrin-B knockout mouse demonstrated long QT and sinus node dysfunction 278 ; however, a more severe and faithful phenotype was reported in heterozygous mice, capturing many of the same observations from humans 279 . KCNE1 knockout mice developed long QT, but only when heart rate deceleration occurred 280 . A transgenic rabbit model overexpressing dominant negative KCNE1-G52R developed long QT and increased susceptibility to drug-induced arrhythmia by accelerating IKs and IKr deactivation kinetics 281 . This model could be useful for examining the proarrhythmic liability of drugs. CACNA1C (Cav 1.2) gainof-function mutations cause LQTS that can be associated with extracardiac manifestations known as Timothy syndrome. In mice, CACNA1C knockout is embryonic lethal, but heterozygous mice survived without any cardiac phenotypes. Cardiac-specific overexpression of the G406R mutation in mice led to long QTc and exerciseinduced PVCs and Torsades de Pointes (TdP) 282 . It was hypothesized that this mutant had altered interaction with AKAP; in fact, when G406R-overexpressing mice were crossed with AKAP150 KO mice, they were protected against all phenotypes of the G406R mutant.
Investigators have also knocked out many of the potassium channel genes in mice to determine their effects on cardiac electrophysiology. When a dominantnegative fragment of KCNA1 was expressed, mice developed LQT and spontaneous ventricular arrhythmias 283 . Interestingly, it was later shown by Glasscock et al. 284 that KCNA1 is preferentially expressed (≈tenfold higher) in atria over ventricles. Programmed electrical stimulation (PES) induced AF in KCNA1 knockout mice but did not lead to any ventricular arrhythmias and no differences in QT interval were observed. Dominant negative overexpression of KCNA5, KCNB1, or KCND2 all prolonged the QT interval without any other overt electrophysiological changes. Mice do not express KCNE3 in the adult heart, but deletion led to long QTc in aged female mice because of hyperaldosteronism 285 .
Limitations-Comments: Mouse models of LQTS should be viewed with caution when the repolarizing current of interest does not reflect the human AP. As transgenic rabbit models become more common, they may find strong ground in advancing our understanding LQT pathogenesis. Rabbits accurately capture sex differences and express a similar repolarization ion channel gene profile as humans. An area of value will be evaluating predisposition to druginduced LQT and arrhythmia 286 , which rabbit models will be best suited to answer. www.nature.com/scientificreports/

Short QT syndrome
Short QT syndrome (SQTS) is an extremely rare disorder; only a few 100 cases have been identified to date. SQTS is caused by the shortening of the cardiac AP. Like long QT syndrome, alterations in cardiac repolarization alter the QT interval. Because of the abbreviated QT interval, the refractory period is also shortened, leaving the heart susceptible to reentrant arrhythmias. Symptoms associated with short QT syndrome include both atrial and ventricular fibrillation, palpitations, and sudden cardiac death. Gain-of-function mutations in KCNH2, KCNQ1, and KCNJ2 or loss-of-function mutations in CACNA1C, CACNB2, CACNA2D1, SCN5A, and SLC4A3 have all been associated with SQTS. There are few genetic animal models available to examine SQTS in vivo. Zebrafish carrying the KCNH2-L499P mutation have a shortened QT interval 287 . Knockdown of SLC4A3 in zebrafish led to a short QT interval that was rescued by expressing WT SLC4A3 but not by SLC4A3-R370H, identified from a patient with SQTS 288 . To appreciably capture the human cardiac AP, a rabbit model was engineered to overexpress KCNH2 carrying the N588K mutation 289 . Transgenic rabbits had shortened AP and QTc but a normal T wave height. Ex vivo perfused hearts had inducible VT and VF; this model is arguably the best available and, as discussed above, rabbit models more accurately typify human cardiac repolarization.

Brugada syndrome
Brugada syndrome (BrS) is a disorder characterized by elevated ST segment, partial bundle branch block, arrhythmia, and sudden cardiac death. It is commonly caused by mutations in SCN5A; however, ≈20 genes are now associated with BrS. Brugada ECG patterns are sometimes observed in overlap syndromes, such as with long QT syndrome, as SCN5A mutations are associated with several arrhythmia disorders. In mice, SCN5A deletion is embryonic lethal, but heterozygous mice survived and developed slowed conduction, pacing-induced VT, and fibrosis with age 290 . Interestingly, the authors observed variability in phenotype penetrance that correlated with NaV1.5 expression levels 291 . A mouse model for an overlap syndrome of LQT and Brugada was generated to carry 1795insD in SCN5A 292 . Homozygous mice were embryonic lethal, but heterozygous mice developed sinus node dysfunction, slowed conduction, bradycardia, and QT prolongation. Interestingly, CaMKII-dependent phosphorylation of wild-type Nav1.5 appears to phenocopy this mouse model, as late current predominates at slower heart rates. Phosphomimetic and phosphoablation mouse models demonstrated that phosphorylation and oxidation modulate Nav1.5 current and susceptibility to arrhythmias 293,294 . A transgenic pig model was generated to better understand BrS disease mechanisms. Pigs were designed to carry the orthologous SCN5AE558X/ + mutation identified in a patient diagnosed with BrS and developed conduction abnormalities and QRS widening but had no elevated ST segment, arrhythmias, or sudden death through 2 years of age 295 . However, ex vivo hearts had increased susceptibility to VF with programmed stimulation. One debate surrounding BrS is whether many of the associated genes cause BrS or simply increase susceptibility to developing BrS. SCN5A mutations have variable and incomplete penetrance and differing phenotypes. Moreover, SCN5A mutations are prevalent in the general population and discerning pathogenesis can be difficult. Given the failure of animal BrS models to reproduce the full clinical syndrome, their utility studying BrS pathogenesis and treatment options remains to be determined.

Genetic arrhythmia syndromes associated with structural heart disease
Animal models have been beneficial for understanding the pathogenesis of arrhythmias caused by mutations in non-ion channel genes that result in structural heart disease. The section below discusses the major arrhythmia syndromes associated with a cardiomyopathy phenotype (ARVC, HCM, and DCM) and microscopic structural disease such as AF, sick sinus syndrome, heart block, and preexcitation syndromes.

Arrhythmogenic right ventricular cardiomyopathy/dysplasia
ARVC/dysplasia is a disease manifesting as fibro-fatty replacement of the right ventricular myocardium and widespread electrophysiological remodeling, predisposing individuals to ventricular arrhythmias and increased risk of sudden death. Approximately half of the ARVC cases are caused by mutations in desmosomal proteins, which make up cell-cell mechanical junctions. Arrhythmias often arise during periods of exercise or stress, suggesting that catecholamines contribute to arrhythmogenesis. Accordingly, one of the primary phenotypes in animal models is catecholamineinduced ventricular arrhythmias. The primary genetic model used to study ARVC has been the mouse. Of note, mice do not get fatty infiltration in the heart, one of the primary phenotypes of ARVC in humans. However, numerous cardiac phenotypes have been found, from remodeling to arrhythmogenesis. The first genetic mouse models to examine the importance of desmosomal proteins were congenital knockouts ( Table 2). Mouse models with global knockout of plakoglobin, desmoplakin, plakophilin-2, and desmoglein-2 are all embryonic lethal. However, heterozygotes survived and developed varying degrees of fibrosis and arrhythmia phenotypes ( Table 2). Subsequent animal models were based on patient mutations and frequently resulted in haploinsufficiency. This seems to be the primary cause, along with repression of the Wnt signaling pathway. DSC2 (desmocollin-2) mutations are rare, and the link between pathogenesis and this protein is not well understood because data are lacking and conflicting. DSC2 knockout mice are viable but did not develop any cardiac phenotypes. However, DSC2 knockdown reportedly led to altered ultrastructure and contractile dysfunction, while overexpression also led to fibrotic remodeling 296 . Germline knockout of DSG2 (desmoglein-2) results in embryonic lethality in mice, but the cardiac-specific knockout led to dilation, fibrosis, and electrophysiological remodeling. Finally, the inhibitor of apoptosis-stimulating protein of p53 has been linked to ARVC; knockout mouse model developed dilation, arrhythmia, and sudden death 297 .
The PKP2 (plakophilin-2) mouse model is well-suited for understanding ARVC mechanisms and disease progression. Heterozygote PKP2 mice survived and developed arrhythmias in the absence of overt structural www.nature.com/scientificreports/ remodeling 296 , which was validated in mice carrying a PKP2 truncation mutant 298 . These findings raised an interesting question about the cause of arrhythmias: how do arrhythmias arise in the absence of structural changes to the heart? Because PKP2 knockout is embryonic lethal, a cardiac-specific inducible PKP2 knockout mouse was generated, which had many of the characteristic ARVC phenotypes: fibrosis, remodeling, reduced ejection fraction, arrhythmia, and sudden death 299 . In this model, PKP2 signaling regulates transcription of many genes involved in Ca2 + homeostasis and proteins of the intracellular Ca2 + release unit are downregulated, causing proarrhythmic RyR2 activity. The authors discovered that flecainide, a class Ic antiarrhythmic that inhibits RyR2 channels, effectively prevented arrhythmias in these mice. Exercise exacerbated RyR2 hyperactivity, and it was shown that membrane-permeable beta-blockers had greater efficacy than nonpermeable beta-blockers 300 . ARVC has been observed in dogs and cats, generally of unknown cause. In these models, fibro-fatty replacement is commonly observed alongside syncope, arrhythmias, and sudden death. Dog models could be considered when disease mechanisms, as they relate to human cardiac (electro)physiology, are essential. Nondesmosomal proteins, such as phospholamban, RyR2, lamin A/C, transmembrane protein 43, and integrin linked kinase have all been associated with ARVC in patients; however, their role is less well understood due to the spectrum of phenotypes in ARVC. For example, some mutations in phospholamban cause DCM, which is a differential diagnosis, but may also present with ARVC. Because of the difficulty of identifying pathogenic mutations, diagnostic criteria rely on interpreting cardiac imaging and ECG data. An important line of investigation understands the molecular mechanisms that drive the development of the disease, as early disease progression escapes detection and the heterogeneity of genes involved makes prognosticating a diagnosis difficult.

Dilated cardiomyopathy
Congenital DCM is characterized by ventricular dilation and is commonly caused by mutations in cytoskeletal or myofibrillar proteins leading to remodeling, reduced ejection fraction, conduction abnormalities, arrhythmia, and sudden death. Many animal models have been created to investigate the structural and functional consequences of these mutations. Here we focus on animal models where arrhythmias are a prominent feature of the reported phenotype (Table 2). Of note, there is significant overlap with the clinical diagnosis of arrhythmogenic (right ventricular) cardiomyopathy.
A widely studied DCM mutant is SCN5A-D1275N. A wide spectrum of phenotypes in different families carrying this mutation have been reported, including AF, conduction defects, and sinus dysrhythmia 301,302 . It is speculated that the variation in genetic background between the families contributes to these phenotypes. Homozygous SCN5A-D1275N mice developed many of the characteristic arrhythmia phenotypes observed in humans, while heterozygous mice did not 303 . Many changes in the ECG parameters reflected a gene dosedependent effect and these features were also observed in zebrafish 304 .
Mutations in LMNA cause severe DCM and sudden death. Several mouse models have been generated that carry mutations identified from patients ( Table 2). A variety of symptoms have been reported and spontaneous arrhythmias occur frequently. Mutations in RMB20 also have a severe phenotype in mice, commonly leading to spontaneous ventricular arrhythmias and sudden death. Duchenne muscular dystrophy has been studied in pig, dog, and mouse models, each having electrophysiological changes ( Table 2).

Hypertrophic cardiomyopathy
HCM is characterized by enlargement of the left ventricle primarily caused by mutations in sarcomeric proteins, leading to increased risk of arrhythmia and sudden death 305 . HCM is commonly caused by mutations in either the beta myosin heavy chain or myosin binding protein C, together accounting for nearly half of all cases. Mice express beta myosin heavy chain during cardiogenesis, but rapidly switch from the beta to the alpha isoform, postnatal. Numerous HCM animal models have been generated; however, the predominant focus has been the study of underlying changes in sarcomere function, contractility, and hypertrophy, without detailed examination of electrophysiological phenotypes. Many studies have reported on arrhythmia susceptibility in ex vivo hearts but only reports examining in vivo arrhythmia phenotypes are discussed here.
Alpha myosin heavy chain mutations are commonly associated with HCM, but the only reports of arrhythmias in an animal model come from the R403Q mutant, which was identified in MYH7 from a human patient. The knock-in mouse model was designed to carry R403Q in MYH6 306 . Mice developed HCM but only had a modest arrhythmia phenotype. A rabbit model was generated to overexpress the transgene but did not exhibit an arrhythmia phenotype 307 . Troponin mutations are also associated with HCM and a transgenic mouse was generated to carry TNNI3-G203S 308 . Mice had conduction defects, but no other arrhythmias. However, when these mice were crossed with MYH6-R403Q mice, they developed a more severe phenotype with conduction defects, LQT, and catecholamine-induced VT. Knockout of MYBPC caused long QT and spontaneous VT in mice 309 .
Several troponin T models have been designed to carry mutations identified in patients. Knollmann et al. 310 showed that TNNT2-I79N mice had tachycardia, isoproterenol-inducible ectopy, and spontaneous nonsustained VT, despite having no hypertrophy or fibrosis. Subsequent work in mice demonstrated that Ca2 +-sensitizing TNNT2 mutations cause inducible arrhythmias, whereas nonsensitizing mutants (R278C) do not leave the heart susceptible to arrhythmias 311 . Myofilament Ca2 + sensitization increases cytosolic Ca2 + -binding affinity, alters int racellular Ca2 + homeostasis, and causes pause-dependent Ca2 + -triggered arrhythmia. In addition, myofilament Ca2 + sensitization causes focal energy deprivation, which further increases arrhythmia susceptibility in mice 312 . Hence, myofilament sensitization per se, caused by drugs, mutations, or posttranslational modifications after myocardial infarction 313 , is a novel arrhythmia mechanism 314 . These reports illustrate the power of murine HCM models for discovering new arrhythmia mechanisms and identifying therapeutic targets. www.nature.com/scientificreports/ Atrial fibrillation AF is the most common arrhythmia and is characterized by rapid, abnormal atrial rhythms, with symptoms manifesting as palpitations, syncope, stroke, and heart failure, among others. The etiology of AF is multifactorial, stemming from environmental factors, diet, lifestyle, family history, medication, and surgery. For an in-depth review of acquired AF and various animal models available, the reader is referred to this review 36 . Many genetic animal models develop AF alongside their primary disease phenotype (see Tables 2 and 3), but the models discussed in this section are more directly related to AF as a primary pathology. One trigger for AF is thought to be hypersensitive and leaky RyR2 channels. Thus, animal models of CPVT (both gain-of-function RyR2 and loss-of-function Casq2) have been used to investigate arrhythmogenic mechanisms and screen therapeutic modalities 315 . It was demonstrated that RyR2 inhibition can attenuate AF in these models. Other CPVT models are susceptible to AF with PES. A more severe phenotype was seen with CREM mice, which developed atrial dilation and spontaneous paroxysmal and persistent AF that worsened with age 316 . A second trigger for AF may be channelopathies that accentuate excitability. KCNE1, SCN5A, KCNQ1, SK2, and SK3 mutations have all been introduced into mice, with varying phenotypes ( Table 2). The KCNE1 knockout mouse develops spontaneous AF 317 . SCN5A models show overt structural changes, fibrosis, conduction abnormalities, and mitochondrial injury. Proteins associated with development, signaling pathways, and transcription regulation have been found to induce AF. Atrial-specific or complete knockout of LKB1 in mice caused electrical and structural remodeling and mice developed spontaneous AF 318,319 . A goat model overexpressing constitutively active TGF-β1 had atrial fibrosis and AF inducibility with PES 320 . Genome-wide association studies have identified many gene loci associated with increased AF risk. AF loci include genes known to affect ion channel function, cardiogenesis, or cell-cell conduction, although in many cases, candidate genes have not been determined. There are several animal models of inherited AF, mostly in mice. A primary challenge with mouse models of AF is that they do not commonly develop spontaneous AF, instead, only uncovering the phenotype with PES. Moreover, AF typically lasts for a brief period (on the order of seconds) before resolving. However, some mouse models have more severe and protracted AF that occurs spontaneously ( Table 2). AF manifests in a wide range of cardiac diseases and resolving causative versus correlative pathogenesis is ongoing for some genetic models. Despite these shortcomings, investigators have successfully captured AF phenotypes in many different mouse models for genes identified in genomewide association studies studies or laboratory testing.
Genome-wide association studies have identified loss of function (PITX2, TBX5, GJA1) and gain of function (KCNN3) variants associated with patients with AF. PITX2 heterozygous mice had normal cardiac parameters except reduced transpulmonary flow; however, ex vivo hearts were more susceptible to atrial pacing-induced arrhythmia 321 . Subsequent work showed AF in vivo with PES and several groups have used this model to study AF and explore treatment 322 , especially since PITX2 is the most common risk locus identified in patients with AF. Inducible deletion of the TBX5 transcription factor led to a much more severe phenotype of spontaneous AF and electrophysiological remodeling within 2 weeks of tamoxifen treatment 323 . Gene profiling identified numerous changes in the expression of Ca2 + handling proteins and ion channels, including PITX2. These data provide a link between TBX5-PITX2 activity and electrophysiological protein regulation in the heart. AF may also be promoted by delayed conduction. Mice carrying the GJA1-G60S/ + mutation are more susceptible to pacing-induced AF 324 . Mice overexpressing KCNN3 had bradyarrhythmias, heart block, abnormal AV node morphology, and sudden death 325 .
Many other models have explored mutations or deletion of other ion channels, transcription factors, developmental pathways, or hormones to examine AF (Table 2). Data from patients have frequently confirmed the downregulation of various proteins, providing yet another link to pathogenesis. AF is a complex multifactorial disease; each animal model is an opportunity to extend our understanding of this disease.

Sick sinus syndrome
Sick sinus syndrome occurs when the sinoatrial node is not capable of generating a normal rhythm. In heterozygous SCN5A ± mice (see also Brugada Syndrome), investigators identified sick sinus syndrome phenotypes and sex differences in sinoatrial node function with age 326 . The hyperpolarization-activated cyclic nucleotide-gated channels (HCN) play an essential role in generating spontaneous pacemaker activity. Various models have examined the deletion of HCN1-4. HCN4 is the most abundant isoform, and congenital deletion in mice is embryonic lethal. However, embryonic hearts had sinus bradycardia 327 . Inducible cardiac-specific knockout led to severe bradycardia, block, and sudden death within days 328 . Mice with germline deletion of HCN1 or HCN2 survived and developed sinus dysrhythmia 329,330 . Deletion of HCN3 did not lead to bradycardia or arrhythmia, but mice had subtle perturbations in their ECG morphology at lower heart rates. Perhaps the most elegant and accurate mouse model of sick sinus syndrome is an inducible HCN4-specific cellular ablation mouse 331 . This model accurately captured many of the phenotypes observed in patients, such as fibrosis of nodal tissue, sinoatrial dysrhythmia, supraventricular tachycardia, VT, and sudden death.

Atrioventricular block
AV block is partial or complete disruption of impulse propagation from the atria to the ventricles. As the single site for collating atrial depolarization and passing it to the ventricle, aberrant AV node function may prevent or delay sinus rhythm from conducting into the ventricle. The AV node makes up part of the conduction system and, as such, expresses many of the same ion channels and gap junctions as the sinoatrial node, bundle of His, and Purkinje cells. AV block is observed in many of the models described above. However, some genes seem to specifically alter AV node conduction and effect block without altering sinus node rhythm. The NKX2-5 gene appears to be definitely related to AV block in patients, and mouse models carrying 2 different mutations independently validated the role of this gene in disease progression 332 www.nature.com/scientificreports/

Preexcitation syndrome
Preexcitation occurs when the ventricles are activated prematurely via an accessory pathway. The most common mouse models generated to study preexcitation syndrome have mutations in PRKAG2, a regulatory subunit of 5′ AMP-activated protein kinase [334][335][336] . Mutations in the TBX2 transcription factor also led to the development of an accessory pathway in mice 337 .

Arrhythmia mechanisms in human induced pluripotent stem cell-derived cardiomyocytes
The advent of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in 2007 has solved one fundamental challenge in basic cardiac research and drug discovery; now, there is an unlimited source of CMs of human origin, both from healthy individuals and patients with cardiac diseases 338 . The hiPSC-CM discovery opened a plethora of different use cases as follows: tests on drug cardiotoxicity and efficacy, understanding arrhythmic mechanisms, modeling diseases in a dish, developing patient-specific models and personalized drugs, and regenerative medicine. Some use cases are in a more developed state than others (Fig. 11). For example, using hiPSC-CMs for drug cardiac safety assessment was theorized in 2013 within the Comprehensive in vitro Proarrhythmia Assay (CiPA) 339 , as a step to confirm the results of in vitro and in silico tests on drug effects on multiple cardiac ion channels and the cardiac action potential (AP). At the time of this review, the CiPA initiative has already obtained the support of regulatory agencies and pharmaceutical companies, and it is at a very advanced state (cipaproject.org/timelines/). Conversely, other applications are still in their infancy, for example, Figure 11. Scope of the review. Although arrhythmogenic properties of hiPSC-CMs are very relevant in all 3 principal application fields (basic research, drug screening, and regenerative therapy), we focus on the first one and on the second one to a lesser extent. Also, we link the hiPSC-based findings to the human/patient context whenever possible. www.nature.com/scientificreports/ the development of personalized therapies informed by in-depth analysis of patient-specific hiPSC-CMs and personalized medicine 340 . Furthermore, the full potential of hiPSC-based applications remains to be harnessed because these cells do not adequately recapitulate (1) morphological and ultrastructural, (2) electrophysiological, (3) contractile, and (4) metabolic properties of native adult human CMs. Efforts aimed at solving these challenges have been recently reviewed by others [341][342][343][344][345] . Here, we review the advances that the hiPSC-based modeling systems have brought so far regarding the understanding of both arrhythmogenic triggers and substrates. First, we assess how different mechanisms of arrhythmogenesis that relate to abnormal impulse formation and propagation have been captured. Then, we explore to what extent can hiPSCbased approaches recapitulate the different atrial and ventricular arrhythmogenic phenotypes. The usability of hiPSC-CMs in investigating drug-induced arrhythmias and sex-dependent arrhythmic characteristics are discussed in the following chapters. Finally, we provide some conclusions and speculate about future directions. One of the aforementioned benefits of hiPSC-CMs is the availability of a pool of in vitro human cell models to reproduce the occurrence of arrhythmias under different conditions, and to understand better the underlying mechanisms 346 , as well as developing pharmacological strategies to reduce them. Examples of use cases are the presence of mutations altering specific membrane channels (channelopathies) [347][348][349][350][351][352][353] , or other cell functions [354][355][356][357][358] , or when administering drugs 359 . For all these use cases, the clear advantage of hiPSC-CMs is that they are from human source. A potential disadvantage is that they are relatively recent in vitro models, and a better understanding of the actual similarity to native human CMs in terms of arrhythmic event development is needed 360 . A well-known scheme to classify mechanisms of cardiac arrhythmias 6,361 identifies 2 macrofamilies, separating nonreentrant and reentrant activities: abnormal impulse formation and improper impulse conduction. In this review, we follow this scheme (Fig. 12), reporting abnormal impulse formation and improper impulse conduction for hiPSC-CMs and expand the mechanisms adding spatial dispersion of repolarization and myocardial heterogeneity (Figs. 11,12,13).

Limitations of hiPSC-CMs
Despite the undeniable value of hiPSC-CMs as a human in vitro model, it is fair to recognize that they suffer specific limitations as models fully representative of hV-CMs, as we reported in the previous sections of this review. In this section, we recapitulate and expand on those limitations (Figs. 11,12,13). www.nature.com/scientificreports/ Some of the ion currents are differently expressed in hiPSC-CMs and hV-CMs. hiPSC-CMs express 2 ion channels that are absent in healthy hV-CMs: If 362 and ICaT 363 . Furthermore, the balance of repolarizing potassium currents is quite different. The expression of IK1 has been reported as low as in human adult atrial CMs (hA-CMs) 364 or even absent 360 . Whereas, the density of IKr is substantially higher in hiPSC-CMs compared with hV-CMs 352,362,365 . The balance of IK1 and If underlies hiPSC-CM automaticity in the voltage clock paradigm; one of the hallmarks of the immature or non-adult hiPSC-CMs. In addition, expression of some ion channels and consequent ion current densities seems to be highly variable in comparison with hV-CMs: lower 366 or similar 362 SCN5A and lower 366 or similar 362 KCNQ1. A huge range of ICaL densities has been reported for hiPSC-CM: 3.3-17.1 pA/pF 367,368 , which is likely because of different methodology. The only study comparing ICaL under identical experimental conditions showed 50% larger densities in hiPSC-CMs versus hV-CMs 363 . Together, the aberrant balance of inward and outward ion currents reduces the repolarization reserve in hiPSC-CMs compared with their native counterparts. For example, Blinova et al. 366 correlate these differences in expressions to the different proarrhythmic effects that a set of drugs (namely, bepridil, ranolazine, and mexiletine) has in hiPSC-CMs. Furthermore, Lemoine et al 362 showed that EHT is much more prone to EADs in comparison with human ventricular tissue.
Ca2 + handling is functional but not fully developed in CMs derived from pluripotent stem cells 369 . Two main points are worth to be mentioned. First, the lack or lower expressions of functional proteins, eg, junctin and triadin (to facilitate RyR function), and of calsequestrin for Ca2 + bufferinas well as lower SERCA and RyR. Second, hiPSC-CMs lack a well-developed T-tubule network that is a hallmark of mature ventricular CMs 370 . This ultrastructural immaturity results in an U-shaped Ca2 + wavefront with a remarkable delay in peak between the cell periphery and center 371 , as observed also in hA-CMs, and a poor excitation-contraction coupling 372 .
The hiPSC-CM contraction is also nonoptimal, not only because of poor excitation-contraction coupling, but also to the immature sarcomeric structure and the orientation in multiple directions of the myofibers within the cell 342 . When compared with native human ventricular tissue, hiPSC-based EHTs lack a positive force-frequency Figure 13. Distinct electrophysiological and ultrastructural characteristics of hiPSC-CMs in comparison with human native/adult ventricular CMs (hV-CMs). Impact of prototypical hiPSC-like ICaL, IK1, and IKr densities, which are 1.5-fold, 0.5-fold, and fourfold in comparison with hV-CM, on the repolarization reserve, simulated as in Ref. 193. The absence of t-tubules in hiPSC-CM results in spatial uncoupling of L-type Ca2 + channel (LTCC) and ryanodine receptors (RyR), as well as irregular distribution of SR Ca2 + handling proteins: RyR, Ca2 + ATPase (SERCA), and its regulatory protein phospholamban (PLN). Furthermore, inositol triphosphate receptors (IP3R) activity is substantially higher in hiPSC-CMs. The panels for were adapted from Ref. 196  www.nature.com/scientificreports/ relation 371 , which is one of the hallmarks of cardiac contractility. Furthermore, the frequency-dependent acceleration of relaxation is much weaker in EHTs 373 . Finally, the hiPSC-CM potential to recapitulate the contractile and remodeling signaling of adult CMs is not fully known. Jung et al. 374 studied the different stages of hiPSCCMs and showed that at day 30 b2-AR signaling is dominant, reflecting a relative immaturity of hiPSC-CMs. By day 60, a transition to downstream signaling by the b1-AR represents a more "adult-like" phenotype. Even by day 90, b1-AR expression does not fully recapitulate the pattern seen in adult human ventricles, and b-ARs only activate CaMKII at this time, although the protein expression reached a maximal level by day 30. The differential maturation of intracellular signaling pathways in hiPSC-CMs should be considered in disease modeling and drug testing.

Emerging etiologies of arrhythmogenesis: opportunity for animal models
Cardiac electrophysiology is regulated not only by the amino acid sequence at the protein level but also by posttranslational modifications, micropeptides, epigenetics, long noncoding RNAs (lncRNA), micro RNAs (miRs), aging, and environmental factors. Unprecedented advances in sequencing technology, deep mutational scanning, and epigenetic and transcriptome mapping have highlighted several new areas of pathogenesis. A primary challenge with many of the ascribed changes is whether they directly cause disease or, rather, follow disease progression. Animal models have been generated to address this challenge and study new areas of arrhythmia biology, with a select few highlighted below. In the past decade, micropeptides have been identified that were previously excluded during annotations of open reading frames due to their small size or position in the transcriptome. In the heart, functionally expressed peptides include examples such as sarcolipin and dwarf open reading frame, which regulate SR Ca reuptake. Sarcolipin protein levels are decreased in humans with AF or heart failure 375 and the sarcolipin knockout mouse developed AF with age 376 . In mice with DCM, overexpression of dwarf open reading frame attenuated the heart failure phenotype, although arrhythmias were not examined 377 . The micropeptides apelin and elabela affect cardiogenesis, fibrosis, hypertrophy, and inotropic responses. Reduced apelin levels predicted major myocardial events and MI scar size in patients with a previous MI event 377 . Apelin knockout mice developed larger scars and increased mortality following MI, although arrhythmias were not described 378 . These findings establish the pathogenic role and therapeutic potential for micropeptides in the heart, but their impact on arrhythmogenesis remains to be examined. Posttranslational modifications, primarily phosphorylation, are documented in many arrhythmias 379 . While phosphorylation by PKC, PKA, and CaMKII are the most studied mediators for changes in Na + channels 293,294,380,381 , Ca2 + channels 382-384 , K + channels 385 , NCX 386 , and RyR2 387 , conflicting data exist regarding the relative contribution of different phosphorylation sites to ion currents. Phosphomimetic and phosphoablation mouse models have been created to tease out the importance of each site to arrhythmogenesis 293,382,388,389 . In addition, posttranslational modification of signaling kinases themselves, including PKA 390 and CaMKII 390 , play a role in arrhythmia susceptibility in response to oxidative stress. Other posttranslational modifications are less well studied, and it remains to be determined whether these cause disease or are merely epiphenomena. Epigenetic DNA modifications regulate protein expression. It is now recognized that many cardiovascular diseases are associated with epigenetic changes. For example, cardiac-specific deletion of HDAC1 and HDAC2 in mice led to dilated cardiomyopathy, arrhythmia, and premature death 391 . A family with a history of autosomal recessive DCM was later discovered to carry homozygous mutation in GATAD1 392 . In AF, epigenetics also contribute to risk as it relates to hypertension, obesity, age, and other factors 393 . Patients with persistent AF have genome-wide changes in DNA methylation 394 , and animal models could help elucidate the pathophysiology of these changes at the whole genome or protein level. MiRs regulate gene expression by RNA complementation and silencing. In the heart, miRs control cardiogenesis and pathways to affect gene expression during development. Some miRs are muscle specific and may serve as macroregulators of ion channel expression. In disease, changes in many different miRs have been documented. In dogs with rapid pacing, nicotine administration reduced miR-133 and miR-590 levels and increased AF risk 395 . Atrial tachypacing in dogs and development of AF was also associated with changes in many miRs compared with control 396 . Dogs with ventricular tachypacing developed CHF and AF and miR-29b levels declined within the first 24 h of pacing 397 . In zebrafish, miR-182 served as a TBX-5 effector and miR-182 upregulation led to block, tachycardia, and arrhythmias 398 . In mice, it was shown that miR-1 directly binds to the C-terminus of Kir2.1 and represses channel activity 399 . Moreover, a single nucleotide polymorphism in miR-1 identified from patients with AF did not suppress Kir2.1 channel activity and failed to rescue arrhythmia inducibility in miR-1 knockdown mice (whereas WT miR-1 did). Of most interest is the therapeutic potential for RNA regulation to reverse disease progression. LncRNA regulates gene expression via several mechanisms and changes in lncRNA expression have been documented in a wide range of diseases. Altered expression of lncRNA has been observed in AF 400 , including altered regulation of PITX2 401 . Another study in rabbits with AF induced by atrial tachypacing purported a mechanism whereby lncRNA "sponges" up miR-328 to regulate CACNA1C 402 . Yet another report indicated changes in lncRNA in AF reduce expression of JP2 and RYR2 403 . These studies demonstrate additional mechanisms of regulation beyond controlling gene expression and open up the field to exciting new opportunities in arrhythmia research.

Conclusion
Antiarrhythmic drugs often work by altering conduction velocity, the length of the refractory period, or both. The properties of the Na + and Ca + 2 channels and the passive electrical properties of cardiac tissue play a role in conduction velocity. The length of the action potential and the length of the refractory period vary greatly between species due to large changes in the K + currents that essentially control repolarization. It is clear that there are species-specific differences in the variables that influence the development of arrhythmias, and it is also clear that no animal model can accurately represent a human with arrhythmias. However, the development of diagnostic and therapeutic methods for supraventricular and ventricular arrhythmias has clearly benefited www.nature.com/scientificreports/ from the knowledge gained from animal experiments. We hope that in the future, new insights will be gained from experiments performed at different scales, including single cells, cell cultures, prepared heart sections, isolated whole hearts, whole hearts from conscious and anesthetized animals, and systems expressing and testing functions of molecules involved in electrical excitation. The development of new diagnostic and treatment methods will result from the integration of these discoveries and not from the use of a single model or experimental approach. Electrophysiological testing should be encouraged in animals with "natural" cardiovascular disease. Animal models have played a key role in advancing our understanding of the mechanisms of human cardiac arrhythmias, but they have also revealed fundamental problems for all forms of disease modelling. In any complex process, it is better to summarize the causal path as much as possible as to empirically model the individual components. The mechanistic insights gained in the last decades illustrate the complexity of the pathogenesis of clinical arrhythmias. Models that integrate the effects of genetic and epigenetic modifiers are needed to analyze the multistep signaling pathways involved, including myocyte heterogeneity, channel processing, and downstream signalling. For decades, animal models have been an essential tool to study arrhythmia pathophysiology and therapeutic approaches. Recent work with genetic mouse models yielded a new diagnostic tool in CRDS, identified lifesaving drug therapies for CPVT, LQT3, and ARVC, identified pathogenic mechanisms, and advanced our understanding of arrhythmogenesis in ways that other models cannot. The broad availability of transgenic mouse models and the option to generate mice with cell-specific and/or time-dependent regulation of gene expression provides a significant advantage of the mouse over other small animal species. A major limitation is that their fast heart rate, small heart size, and differential ionic currents do not fully recapitulate human cardiac electrophysiology. While guinea pigs and rabbits can overcome some of these electrophysiological limitations, genetic manipulation is limited in these species. Large animal electrophysiological and hemodynamic parameters match humans more closely but can be prohibitively expensive. Regardless, large animals are useful for preclinical studies evaluating new drugs, gene therapy, and devices that necessitate large animal models before moving to humans. Here, we provide an in-depth review of existing animal models to help interpreting published arrhythmia mechanisms as well as planning future experimental studies investigating cardiac arrhythmia diseases.
In this review, we showed how hiPSC-CMs can recapitulate arrhythmia mechanisms observed in adult CMs, such as abnormal impulse formation and conduction abnormalities, as well as disease phenotypes because of channelopathies (eg, LQT syndrome) or cardiomyopathies (eg, HCM and DCM) that trigger arrhythmic behaviors. However, it can be argued that thus far they have provided only limited new insights into arrhythmia mechanism.
In summary, animal models remain of crucial importance for the discovery and development of cardiac antiarrhythmic drugs. These models help evaluate drug candidates, predict therapeutic efficacy and safety, and better understand the underlying processes of arrhythmogenesis. Future research on effective therapies to prevent and treat cardiac arrhythmias is promising as animal models are improved and more commonly used in conjunction with complementary methods.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.